A sheath flow gating interface for the on-line coupling of solid-phase extraction with capillary electrophoresis.

A sheath flow gating interface (SFGI) is presented for the on-line coupling of solid-phase extraction (SPE) with capillary electrophoresis (CE). The design, construction and operation of the SFGI are described in detail. After operating conditions were investigated and selected, the SFGI was evaluated on a SPE-CE-UV setup using hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith as the absorbent and using three phenols as the test analytes. The preconcentration factors obtained with the SPE-CE-UV system and the SPE-UV part are 530 and 550, respectively. The plate numbers obtained using the SPE-CE-UV system are slightly better than or comparable to those with the CE-UV part. The precisions (RSDs) of 100 consecutive injections are 2.43%, 3.86%, and 4.25% for peak height, peak area and migration time, respectively. The measured recoveries for the river water samples spiked at three different levels are in the range of 93.6-102.8% with the interday RSD values ranging from 2.0 to 4.5% (n=3). These data collectively demonstrate that the SFGI has the ability to exactly and reproducibly transfer nanoliters of fractions from SPE onto CE with no degradation of the efficiencies of SPE and CE, suggesting a great potential to be routinely used for the coupling of SPE, microcolumn LC or FIA with CE.

A miniaturized transverse flow gating interface for the on-line coupling of solid-phase extraction with capillary electrophoresis.

A miniaturized transverse flow gating interface (µ-TFGI) is presented for the on-line coupling of solid-phase extraction with capillary electrophoresis (SPE-CE). The fabrication and operation procedures of the µ-TFGI are described in detail. After the operation conditions were investigated and selected, the µ-TFGI was evaluated on an SPE-CE-UV setup using clenbuterol (CLB) as the test analyte. The preconcentration factors obtained with the SPE-CE-UV system and the SPE-UV part were 600 and 620, respectively. The plate numbers obtained within 3 min were 100000 and 80000 for automatic injection via the µ-TFGI and manual direct injection, respectively. The precisions were 2.4 - 6.8% (RSD, %, n = 9) and 3.1% (RSD, %, n = 27) for the recovery (94.3 - 101.9%) and migration time of CLB spiked in urine samples, respectively. These results demonstrate that the µ-TFGI has the ability to exactly and reproducibly transfer nanoliters of fractions from SPE onto CE with no degradation of the efficiencies of SPE and CE.

Silica-particle-supported zwitterionic polymer monolith for microcolumn liquid chromatography.

A silica-particle-supported zwitterionic polymeric monolithic column, shortened as supported column (S-column), was prepared by the in situ polymerization of methacrylic acid, ethylene dimethacrylate, and 2-(dimethylamino)ethyl methacrylate in the presence of a ternary porogenic solvent containing water, methanol, and cyclohexanol in a 250 µm id fused-silica capillary prepacked with 5 µm bare silica particles. In the S-column, a thin layer of the polymers was formed around the silica particles in the form of nanoglobules, leaving the interstitial spaces between the particles free for liquid flow. The effects of the preparation conditions on the morphology of the monolith were investigated by scanning electron microscopy and backpressure measurements. The selected volumetric ratio of porogens, monomer concentration, polymerization time, and temperature are 1:1:8 (water/methanol/cyclohexanol), 25% v/v, 5 h, and 60°C, respectively. The S-column was evaluated by comparison with its conventional organic counterpart in terms of morphology, mechanical stability, permeability, swelling-shrinking behavior, capacity, and efficiency. The results demonstrate that the S-column is superior to its counterpart in all the terms with the exception of permeability. The above merits and zwitterionic property of the S-column were further confirmed by separate separations of four inorganic anions and three organic cations.

N'-(3-Bromo-5-chloro-2-hydroxy-benzyl-idene)-2-chloro-benzohydrazide methanol solvate.

In the title compound, C(14)H(9)BrCl(2)N(2)O(2)·CH(4)O, the dihedral angle between the two benzene rings is 49.2 (2)° and an intra-molecular O-H⋯N hydrogen bond occurs. In the crystal struture, mol-ecules are linked by O-H⋯O and N-H⋯O hydrogen bonds.

2-Chloro-N'-(2-hydr-oxy-4-methoxy-benzyl-idene)benzohydrazide.

In the title compound, C(15)H(13)ClN(2)O(3), the dihedral angle between the two benzene rings is 82.09 (10)° and an intra-molecular O-H⋯N hydrogen bond occurs. In the crystal structure, N-H⋯O hydrogen bonds link mol-ecules into chains propagating in [100].

Capillary electrophoresis of gene mutation.

This chapter illustrates the usefulness of capillary electrophoresis (CE) for the detection of gene mutation, i.e., point mutation, methylation, and microsatellite analysis. In order to provide a general description of the main results and challenges in the field, some relevant applications and reviews on CE of gene mutation are tabulated. Furthermore, some detailed experimental procedures are shown. Several CE methods of gene mutation detection were developed including the following: (1) single-strand conformation polymorphism with capillary electrophoresis; (2) SNaPshot analysis; (3) constant denaturant capillary electrophoresis; (4) microsatellite analysis; and (5) methylation analysis.

Capillary electrophoresis of oxidative DNA damage.

Urinary 8-hydroxy-2'-deoxyguanosine (8OHdG) is an excellent marker of oxidative DNA damage. Until now, urinary 8OHdG has been measured by high-performance liquid chromatography with electrochemical detection. A simple and sensitive method for the analysis of urinary 8OHdG by capillary electrophoresis with end-column amperometric detection has been developed and is described in this chapter. A single-step solid-phase extraction procedure was optimized and used for extracting 8OHdG from human urine. To improve the sensitivity of this method, a new focusing technique based on a dynamic pH junction was used. In the end, the urinary concentration of 8OHdG in healthy persons, patients with cancer, patients with diabetic nephropathy, and smokers was determined. Emphasis is focused on the establishment and application of the methods.

[Separation and determination of oleanolic acid and ursolic acid from Cornus officinalis by capillary electrophoresis].

A cycledextrin-modified micellar electrokinetic chromatography technique for separating and determination oleanolic acid and ursolic acid in the fruit of Cornus officinalis was developed. The two bio-active components was completely separated using a buffer containing 60 mmol/L SDS, 2.5 mmol/L NaH2PO4, 2.5 mmol/L Na2B4O7, 8 mmol/L DM beta-CD,8 mmol/L beta-CD and 30% (v/v) 2-propanol. The correlation cofficients of the linear calibration graphs for oleanolic acid and ursolic acid are 0. 9996 and 0.9995, respectively. The effects of SDS, 2-propanol, dimethyl-beta-cyclodextrin, beta-cyclodextrin, NaH2PO4, Na2B4O7 and pH on separation were investigated.

Determination of monoamines in urine by capillary electrophoresis with field-amplified sample stacking and amperometric detection.

A simple, rapid and low-cost method using capillary electrophoresis coupled with field-amplified sample stacking and electrochemical detection was developed for the separation and determination of monoamines. In this present work, a systematic study of the parameters (pH value and concentration of electrophoretic buffer, composition of sample solvent, injection voltage and time) affecting separation and on-line concentration of monoamines has been performed enabling the detection sensitivity of these monoamines to be improved by 5,000 times compared with the conventional electrokinetic injection. This developed method was applied to the direct analysis of these monoamines in human urine without off-line sample preconcentration. Due to the requirement for urine dilution to minimize the detrimental effects of high salt on analyte stacking, the real sensitivity improvement is about 50-fold when applying the optimized method to urine samples. In order to quantitate these monoamines accurately, internal standard calibration curves were constructed with standard monoamines in presence of salt with similar concentration as in human urine. In the method validation, the calibration curves were linear over a range of 1.0 x 10(-9) to 2.5 x 10(-8) mol/L for each monoamine and the limits of detection (signal to noise ratio of 3) for these monoamines were in the sub-nmol/L concentration range (6.0 x 10(-10) mol/L).

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.

[Determination of dopamine and norepinephrine in Portulaca oleracea L. by micellar electrokinetic capillary chromatography with amperometric detection].

A method based on micellar electrokinetic capillary chromatography with electrochemical detection (MEKC-EC) was developed for the simultaneous determination of norepinephrine (NE) and dopamine (DA) in Chinese herbal plant extracts of Portulaca oleracea L. The effects of several factors such as the acidity and concentration of running buffer, sodium dodecylsulfate (SDS) concentration and detection potential were investigated to acquire the optimum conditions. The two analytes could be well separated within 12 min in a 64 cm capillary at the separation voltage of 25 kV in a 10 mmol/L phosphate buffer (pH 6.23) containing 10 mmol/L SDS. The excellent linearity was obtained in the concentration range from 1.0 x 10(-6) to 5.0 x 10(-4) mol/L. The detection limits of concentration were 4.2 x 10(-7) mol/L for NE and 8.7 x 10(-7) mol/L for DA and those of quantity were 0.41 fmol for NE and 1.45 fmol for DA at a signal-to-noise ratio of 3. This method was successfully used in the analysis of Portulaca oleracea L. without derivatization procedure, and real average contents of NE and DA in Portulaca oleracea L. were 0.015% and 0.20%, respectively.

Fluorescent-based single-strand conformation polymorphism/heteroduplex capillary electrophoretic mutation analysis of the P53 gene.

Fluorescent-based single-strand conformation polymorphism (F-SSCP) analysis with capillary electrophoresis (CE) is the most common method for the detection of mutation because of its high sensitivity and resolution. In this study, we prepared an inexpensive linear polyacrylamide (LPA), and successfully applied it to CE-SSCP analysis and tandem CE-SSCP/heteroduplex analysis (HA) of the P53 gene on an ABI capillary genetic analyzer. A comparison of the sieving capabilities of a homemade LPA and commercial polydimethylacrylamide (PDMA) demonstrates that the homemade LPA has a higher resolution, a shorter analysis time, and is more suitable for tandem SSCP/HA than commercial PDMA. To show the usefulness, mutations of P53 gene exon 7 - 8 in 37 tumor samples were investigated by using homemade LPA. The results indicate that 10 mutations were found in 9 of 37 cases; the majority of P53 mutations were missense mutations, and 70% were located in exon 7, which plays an important role in neoplastic progression in human tumorigenesis.

[Determination of curcumin in urine by capillary electrophoresis].

The major component of the plant curcuma longa (a widely cultivated tropical plant in Asia and Central America) is curcumin. Curcumin has been reported to have very strong anti-inflammatory, anti-carcinogenic, anti-oxidant, antiallergic, anti-bacterial, and anti-tumor activities. Little is known about the absorption, distribution, and metabolism of curcumin in human beings. The first step in in vivo physiological and pharmacokinetic studies is to develop a method to measure curcumin in body fluid. A rapid capillary electrophoretic (CE) method with diode array detection was established for the determination of curcumin in human urine. It could be rapidly determined within 2.5 min. The optimized experimental conditions were as follows: 15 mmol/L Na2B4O7 as buffer, applied voltage 20 kV, temperature 25 degrees C and detection wavelength 262 nm. The method has been validated and shows good performance with respect to selectivity, reproducibility and limit of detection. Curcumin had good linearity in the range of 10 - 300 mg/L, and the recoveries of curcumin added in urine were more than 96.3% with relative standard deviations (RSDs) less than 2.3%. The method is sensitive, fast and accurate and can be used to determine curcumin in urine.

Determination of urinary oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine and the association with cigarette smoking.

8-hydroxy-2'-deoxyguanosine (8OHdG) has been widely used as a biomarker of oxidative DNA damage in both animal models and human studies. To evaluate the effect of cigarette smoking on oxidative stress, we studied the levels of urinary 8OHdG from smokers and non-smokers and investigated the association with cigarette smoking. The urinary 8OHdG concentrations were determinated by capillary electrophoresis with end-column amprometric detection (CE-AD) after a single-step solid phase extraction (SPE), and then quantitatively expressed as a function of creatinine excretion. To increase the concentration sensitivity, a dynamic pH junction was used and the focusing effect was obvious when using 30mM phosphate (pH 6.50) as sample matrix. The limit of detection is 4.3nM (signal-to-noise ratio S/N=3). The relative standard deviation (R.S.D.) was 1.1% for peak current, and 2.3% for migration time. Based on the selected CE-AD method, it was found that the mean value of urinary 8OHdG levels in the smokers significantly higher than that in non-smokers (31.4+/-18.9 nM versus 14.4+/-7.6 nM , P=0.0004; 23.5+/-21.3 mug g (-)(1) creatinine versus 12.6+/-13.2 mug g (-)(1) creatinine, P=0.028).

Measurement of histamine in individual rat peritoneal mast cells by capillary zone electrophoresis with electrochemical detection.

Capillary zone electrophoresis was employed for the analysis of histamine in single rat peritoneal mast cells using an amperometric detector with a carbon fiber microdisk bundle electrode. In this method, individual mast cells and then 0.02 mol/l NaOH as a lysing solution are injected into the front end of the separation capillary by electromigration with an aid of a inverted microscope. A cell injector was constructed. Using it, the cell suspension was static, when a voltage for injecting single cells was applied. Histamine in single rat peritoneal mast cells have been identified. Quantitation has been accomplished through the use of calibration curves. The mean amount of histamine for nine cells is 95.8 fmol, which is consistent with the literature value.

Carbon fiber bundle-Au-Hg dual-electrode detection for capillary electrophoresis.

The assembly of a dual-electrode amperometric detection system for capillary electrophoresis is developed. The dual-electrode detector consists of different kinds of electrode material (carbon fiber and Au-Hg). The dual-electrode is placed on the outlet end of the separation capillary. Cysteine, glutathione, ascorbic acid and uric acid can be detected simultaneously and selectively at the two electrodes of the dual electrode, respectively. The capillary electrophoresis-dual-electrode detection system has be used to determine these compounds in human blood samples.