Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same speciticity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients. (C) 2004 Elsevier B.V. All rights reserved.

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mm borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic N134 cells induced by arsenic trioxide (AS(2)O(3)) at low concentration (1-2 mu m). Buthionine sulfoximine (BSO), in combination with AS(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of AS(2)O(3) and hydrogen peroxide (H2O2) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.

Determination of dissociation constants of Strychnos alkaloids from Strychnos nux-vomica L. by capillary electrophoresis

An accurate capillary electrophoresis method was developed for the determination of dissociation constants of five Strychnos alkaloids from Strychnos nux-vomica L. The method relies on measuring the effective mobility of the solute as a function of the buffer pH. The mathematical relationship was strictly derived from the fundamental electrophoresis theory and the dissociation equilibrium of a weak base without any simplifications. Careful optimization of the running buffer permitted base-line resolution of the five structurally similar alkaloids.

Determination of heat capacities and thermodynamic properties of 2-(chloromethylthio)benzothiazole by an adiabatic calorimeter

The molar heat capacities of 2-(chloromethylthio)benzothiazole (molecular formula C8H6ClNS2, CA registry no. 28908-00-1) were measured with an adiabatic calorimeter in the temperature range between (80 and 350) K. The construction and procedures of the calorimeter were described in detail. The performance of the calorimetric apparatus was evaluated by heat capacity measurements on alpha-Al2O3. The deviation of experiment heat capacities from the corresponding smoothed values lies within 0.3%, whereas the uncertainty is within +/-0.5%, compared with that of the recommended reference data over the whole experimental temperature range. A fusion transition was found from the C-p-T curve of 2-(chloromethylthio)benzothiazole. The melting temperature and the molar enthalpy and entropy of fusion of the compound were determined to be T-m = (315.11 +/- 0.04) K, Delta(fus)H(m) = (17.02 +/- 0.03) kJ(.)mol(-1), and Delta(fus)S(m) = (54.04 +/- 0.05) J(.)mol(-1.)K(-1), respectively. The thermodynamic functions (H-T - H-298.15) and (S-T - S-298.15) were also derived from the heat capacity data. The molar fraction purity of the 2-(chloromethylthio)benzothiazole sample used in the present calorimetric study was determined to be 99.21 by fraction melting.

Application of capillary zone electrophoresis in the separation and determination of the curcuminoids in urine

The major components of the plant curcuma longa are the curcuminoids that include curcumin, demethoxycurcumin and bisdemethoxycurcumin. It has been reported the curcuminoids have some important activities. A new CZE method with diode array detection has been developed for the separation and determination of the curcumin, demethoxycurcumin and bisdemethoxycurcumin. Three curcuminoids could be readily separated within 7 min with a 15 mM sodium tetraborate buffer containing 10% methanol (v/v) at pH 10.8, 25 kV and 30 degrees C. The method has been validated and shows good performance with respect to selectivity, reproducibility, linearity, limits of detection and recovery. The proposed method was successfully applied to determine the curcuminoids in urine. (c) 2004 Elsevier B.V. All rights reserved.

Determination of urinary nucleosides by direct injection and coupled-column high-performance liquid chromatography

A coupled-column liquid chromatographic method for the direct analysis of 14 urinary nucleosides is described. Efficient on-line clean-up and concentration of 14 nucleosides from urine samples were obtained by using a boronic acid-substituted silica column (40 turn x 4.0 mm I.D.) as the first column (Col-1) and a Hypersil ODS2 column (250 mm x 4.6 mm I.D.) as the second column (Col-2). The mobile phases applied consisted of 0.25 mol/L ammonium acetate (pH 8.5) on Col-1, and of 25 mmol/L potassium dihydrogen phosphate (pH 4.5) on Col-2, respectively. Determination of urinary nucleosides was performed on Col-2 column by using a linear gradient elution comprising 25 mmol/L potassium dihydrogen phosphate (pH 4.5) and methanol-water (60:40, v/v) with UV detection at 260 nm. Urinary nucleosides analysis can be carried out by this procedure in 50 min requiring only pH adjustment and the protein precipitation by centrifugation of urine samples. Calibration plots of 14 standard nucleosides showed excellent linearity (r > 0.995) and the limits of detection were at micromolar levels. Both of intra- and inter-day precisions of the method were better than 6.6% for direct determination of 14 nucleosides. The validated method was applied to quantify 14 nucleosides in 20 normal urines to establish reference ranges. (c) 2005 Elsevier B.V. All rights reserved.

Quantitative determination of compounds in tobacco essential oils by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

A quantitative analysis of the individual compounds in tobacco essential oils is performed by comprehensive two-dimensional gas chromatography (GC x GC) combined with flame ionization detector (FID). A time-of-flight mass spectrometer (TOF/MS) was coupled to GC x GC for the identification of the resolved peaks. The response of a flame ionization detector to different compound classes was calibrated using multiple internal standards. In total, 172 compounds were identified with good match and 61 compounds with high probability value were reliably quantified. For comparative purposes, the essential oil sample was also quantified by one-dimensional gas chromatography-mass spectrometry (GC/MS) with multiple internal standards method. The results showed that there was close agreement between the two analysis methods when the peak purity and match quality in one-dimensional GC/MS are high enough. (c) 2005 Elsevier B.V. All rights reserved.

Determination of ionic resistance and optimal composition in the anodic catalyst layers of DMFC using AC impedance

In the present study, a method based on transmission-line mode for a porous electrode was used to measure the ionic resistance of the anode catalyst layer under in situ fuel cell operation condition. The influence of Nafion content and catalyst loading in the anode catalyst layer on the methanol electro-oxidation and direct methanol fuel cell (DMFC) performance based on unsupported Pt-Ru black was investigated by using the AC impedance method. The optimal Nafion content was found to be 15 wt% at 75 degrees C. The optimal Pt-Ru loading is related to the operating temperature, for example, about 2.0 mg/cm(2) for 75-90 degrees C, 3.0 mg/cm2 for 50 degrees C. Over these values, the cell performance decreased due to the increases in ohmic and mass transfer resistances. It was found that the peak power density obtained was 217 mW/cm(2) with optimal catalyst and Nafion loading at 75 degrees C using oxygen. (c) 2005 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.

Determination of organophosphorus pesticides in cucumber and potato by stir bar sorptive extraction

Organophosphorus pesticides (OPPs) in vegetables were determined by stir bar sorptive extraction (SBSE) and capillary gas chromatography with thermionic specific detection (TSD). Hydroxy-terminated polydimethylsioxane (PDMS) prepared by sol-gel method was used as extraction phase. The effects of extraction temperature, salting out, extraction time on extraction efficiency were studied. The detection limits of OPPs in water were <= 1.2 ng/l. This method was also applied to the analysis of OPPs in vegetable samples and matrix effect was studied. Linear ranges of OPPs in vegetable samples were 0.05-50 ng/g with detection limits <= 0. 15 ng/g and the repeatability of the method was less than 20% relative standard deviation. (c) 2005 Elsevier B.V. All rights reserved.

Development and application of a sol-gel immunosorbent-based method for the determination of isoproturon in surface water

An immunosorbent was fabricated by encapsulation Of monoclonal anti-isoproturon antibodies in sol-gel matrix. The immunosorbent-based loading, rinsing and eluting processes were optimized. Based on these optimizations, the sol-gel immunosorbent (SG-IS) selectively extracted isoproturon from an artificial mixture of 68 pesticides. In addition to this high selectivity, the SG-IS proved to be reusable. The SG-IS was combined with liquid chromatography-tandem mass spectrometry (LC-MS-MS) to determine isoproturon in surface water, and the linear range was up to 2.2 mu g/l with correlation coefficient higher than 0.99 and relative standard deviation (RSD) lower than 5% (n = 8). The limit of quantitation (LOQ) for 25-ml surface water sample was 5 ng/l. (c) 2006 Elsevier B.V. All rights reserved.

Determination of heavy metal ions by capillary electrophoresis with contactless conductivity detection after field-amplified sample injection

A method has been developed for determining of heavy metal ions by field-amplified sample injection capillary electrophoresis with contactless conductivity detection. The effects of the 2-N-morpholinoethanesulfonic acid/histidine (MES/His) concentration in the sample matrix, the injection time and organic additives on the enrichment factor were studied. The results showed that MES/His with a low concentration in the sample matrix, an increase of the injection time and the addition of acetonitrile improved the enrichment factor. Four heavy metal ions (Zn2+, Co2+, Cu2+ and Ni2+) were dissolved in deionized water, separated in a 10 mM MES/His running buffer at pH 4.9 and detected by contactless conductivity detection. The detection sensitivity was enhanced by about three orders of magnitude with respect to the non-stacking injection mode. The limits of detection were in the range from 5 nM (Zn2+) to 30 nM (Cu2+). The method has been used to determine heavy metal ions in tap water.