Liu-Yin Fan

Separation and determination of abused drugs clenbuterol and salbutamol from complex extractants in swine feed by capillary zone electrophoresis with simple pretreatment

A simple but efficient capillary zone electrophoresis (CZE) method was developed for the fast separation and determination of two misused beta2-agonists clenbuterol (CLB) and salbutamol (SAL) from complex background extractants existing in swine feed samples. The proper experimental conditions were achieved as 20.0 mmol/l pH 10.5 Na(2)HPO(4)-NaOH buffer, 20 kV applied voltage, fused-silica capillary of 60.5 cm x 75 microm i.d. (50 cm to detector). Under the proper conditions, the two abused drugs can be online isolated from the complex extractants and the separation between CLB and SAL is good, all of the target analytes can be detected within 4.5 min. The linear response of CLB and SAL concentration ranges from 2.0 to 100.0 microg/ml with high correlation coefficient (R(2)=0.9990) and (R(2)=0.9986), respectively. Limit of detection (LOD) and limit of quantification (LOQ) was calculated to be 0.95 and 3.17 microg/ml for CLB, 1.07 and 3.57 microg/ml for SAL. The precision values (expressed as R.S.D.) of intra- and inter-day were 1.24-2.36% and 0.90-3.85% for CLB, 0.47-1.64% and 0.91-3.46% for SAL. Recoveries spiked at levels 5.0, 25.0, 80.0 microg/ml ranged between 93.30% and 104.33% with R.S.D. less than 5%. Finally, the developed method has been applied to the analysis of real swine feed samples and has achieved satisfactory results.

Differential Proteomic Analysis of Human Saliva using Tandem Mass Tags Quantification for Gastric Cancer Detection

Novel biomarkers and non-invasive diagnostic methods are urgently needed for the screening of gastric cancer to reduce its high mortality. We employed quantitative proteomics approach to develop discriminatory biomarker signatures from human saliva for the detection of gastric cancer. Salivary proteins were analyzed and compared between gastric cancer patients and matched control subjects by using tandem mass tags (TMT) technology. More than 500 proteins were identified with quantification, and 48 of them showed significant difference expression (p < 0.05) between normal controls and gastric cancer patients, including 7 up-regulated proteins and 41 down-regulated proteins. Five proteins were selected for initial verification by ELISA and three were successfully verified, namely cystatin B (CSTB), triosephosphate isomerase (TPI1), and deleted in malignant brain tumors 1 protein (DMBT1). All three proteins could differentiate gastric cancer patients from normal control subjects, dramatically (p < 0.05). The combination of these three biomarkers could reach 85% sensitivity and 80% specificity for the detection of gastric cancer with accuracy of 0.93. This study provides the proof of concept of salivary biomarkers for the non-invasive detection of gastric cancer. It is highly encouraging to turn these biomarkers into an applicable clinical test after large scale validation.

Quantitative investigations on moving chelation boundary within a continuous EDTA-based sample sweeping system in capillary electrophoresis.

Recently, the EDTA‐based sweeping of metal ion that combines chelate formation and in‐line sample pre‐concentration was advanced. However, the moving chelation boundary (MCB) existing in the sweeping system has not been investigated for the quantitative illumination of the mechanism of metal ion sweeping yet. In this paper, the model of MCB was developed from the EDTA‐based sweeping. The theoretical studies were performed for boundary movements and controllable sweeping of metal ion. The theoretical studies show that the sweeping system contains the MCB boundary and chelate boundary, and the difference between the MCB and chelate boundary velocities plays a key role in the stacking efficiency of metal ion. To demonstrate the validity of the theoretical conclusion, the experiments of continuous EDTA‐based sweeping were performed with CE and a home‐made apparatus of a large tube. The home‐made apparatus consisted of a glass tube used for the run of continuous EDTA‐based sweeping and two peristaltic pumps used for the supply of solution. The experimental results were in accordance with the theoretical conclusions above. The developed theory holds evident for significance for the mechanism of controllable stacking of metal ions in EDTA‐based sweeping system in CE.

Controlling of band width, resolution and sample loading by injection system in a simple preparative free-flow electrophoresis with gratis gravity

In this paper, the controllable band width, resolution and sample loading were investigated by the injection system of free-flow electrophoresis (FFE) with gratis gravity. Two general injection methods were described herein. The first method was the one in which sample injection fluxes were variable with constant background flux, while the second was the one in which the background fluxes were flexible with stable sample flux. With methyl green and crystal violet as two viewable model compounds, a series of experiments were performed, and the experimental results revealed that (1) the sample band width could be under desiring control through the regulation of ratios between sample and background fluxes, (2) the separative resolution could be also adjusted elaborately via the regulation of flux ratios during the separation of methyl green and crystal violet with only one charge disparity, and (3) the sample loading could be conveniently controlled via the flux ratios and an approximate maximum sample loading could be selected under the condition of just completed separation of two adjoining solutes. In addition, it was observed that the flux ratio had soft influence on the separative resolution of two solutes. These results were of significance to the designs on band width, resolution and sample loading in the newly developed FFE device with gratis gravity.

Separation and determination of acrylamide in potato chips by micellar electrokinetic capillary chromatography.

A simple and rapid method using micellar electrokinetic capillary chromatography (MEKC) was developed for the separation and determination of acrylamide in potato chips at low levels for the first time. The experimental conditions for the separation and quantification of acrylamide were optimized at first. The optimized conditions were: 50mmol/L Na(2)B(4)O(7) and 40mmol/L SDS at pH 10.0, 12kV applied voltage, 76cm total length (67cm effective length) and 75mum i.d. capillary, 198nm wavelength, 15cm high 25s hydrodynamics sample injection, 20 degrees C air-cooling. The linear response of acrylamide concentration ranges from 0.5 to 100mug/mL with high correlation coefficient (r=0.9986, n=9). The LOD and LOQ were estimated to be 0.1 and 0.33mug/mL based on S/N=3 and 10. The precision values (expressed as R.S.D.) of intra- and inter-day were 0.86-4.35% and 2.61-9.65%, respectively. Recoveries spiked at levels 2, 20, 60mug/mL ranged between 90.86% and 99.6% with R.S.D. less than 6.5%. Finally, the developed method has been applied to the analysis of real samples and has achieved satisfactory results. All of these indicated that it was a reliable method for the quantification of acrylamide in potato chips.

Simple boric acid-based fluorescent focusing for sensing of glucose and glycoprotein via multipath moving supramolecular boundary electrophoresis chip.

Boric acid-based fluorescent complex probe of BBV-HPTS (boronic acid-based benzyl viologen (BBV) and hydroxypyrene trisulfonic acid trisodium salt (HPTS)) was rarely used for sensitive sensing of saccharide (especially glycoprotein) via electrophoresis. We proposed a novel model of moving supramolecular boundary (MSB) formed with monosaccharide or glycoprotein in microcolumn and the complex probe of BBV-HPTS in the cathodic injection tube, developed a method of MSB fluorescent focusing for sensitive recognition of monosaccharide and glycoprotein, and designed a special multipath capillary electrophoresis (CE) chip for relative experiments. As a proof of concept, glucose and hemoglobin A1c (HbA1c) were respectively used as the mode saccharide and glycoprotein for the relevant demonstration. The experiments revealed that (i) the complex of BBV-HPTS could interact with free glucose or bound one in glycoprotein; (ii) the fluorescent signal was a function of glucose or glycoprotein content approximately; and (iii) interestingly the fluorescent band motion was dependent on glucose content. The developed method had the following merits: (i) low cost; (ii) low limit of detection (down to 1.39 pg/mL for glucose and 2.0 pg per capillary HbA1c); and (iii) high throughput (up to 12 runs or more per patch) and speed (less than 5 min). The developed method has potential use for sensitive monitoring of monosaccharide and glycoprotein in biomedical samples.

Fast and selective determination of total protein in milk powder via titration of moving reaction boundary electrophoresis

In this paper, moving reaction boundary titration (MRBT) was developed for rapid and accurate quantification of total protein in infant milk powder, from the concept of moving reaction boundary (MRB) electrophoresis. In the method, the MRB was formed by the hydroxide ions and the acidic residues of milk proteins immobilized via cross‐linked polyacrylamide gel (PAG), an acid‐base indicator was used to denote the boundary motion. As a proof of concept, we chose five brands of infant milk powders to study the feasibility of MRBT method. The calibration curve of MRB velocity versus logarithmic total protein content of infant milk powder sample was established based on the visual signal of MRB motion as a function of logarithmic milk protein content. Weak influence of nonprotein nitrogen (NPN) reagents (e.g., melamine and urea) on MRBT method was observed, due to the fact that MRB was formed with hydroxide ions and the acidic residues of captured milk proteins, rather than the alkaline residues or the NPN reagents added. The total protein contents in infant milk powder samples detected via the MRBT method were in good agreement with those achieved by the classic Kjeldahl method. In addition, the developed method had much faster measuring speed compared with the Kjeldahl method.

Mid-scale free-flow electrophoresis with gravity-induced uniform flow of background buffer in chamber for the separation of cells and proteins

A large-scale free-flow electrophoresis (LS-FFE) is often too large for cell separation of lab scale, whereas micro-FFE (μFFE) has great difficulty in cell isolation due to easy blockage by cell accumulation in μFFE. In this study, a mid-scale FFE (MS-FFE) is developed for cell and protein separations. The volume of the separation chamber (70×40×0.1-0.8 mm) is from 280 μL to 2.24 mL, much lower than that in an LS-FFE but higher than that in a μFFE. Gravity is used for uniform flow of the background buffer only via a single pump with 16 channels and the sample is injected via an adjuster originally used for clinical intravenous injection. The experiments reveal that the hydrodynamic and electrohydrodynamic flows are much stable, and the Joule heat can be effectively dispersed without obvious positive or negative deviation as shown by the omega plots. By the device, Escherichia coli and Staphylococcus aureus, which easily accumulate to block μFFE and are separated with difficulty due to their same negative charges carried, can be well isolated under the conditions of 4.5 mM pH 8.5 Tris-boric buffer (4.5 mM Tris, 4.5 mM boric acid) with 0.10 mM ethylene diamine tetraacetic acid and 5% m/v sucrose, 200 μL/min, 800 V, and sample injection via inlet 4. The mid-scale FFE device could also be used for the separation of three model proteins of horse heart cytochrome c, myoglobin and bovine serum albumin. The device has clear significance for mid-scale separation of cells and proteins.

Sensitive Determination of Barbiturates in Biological Matrix by Capillary Electrophoresis Using Online Large-Volume Sample Stacking*

Abstract:  In China, some forensic cases are caused by barbiturates. Thus, the determination of trace level barbiturates in body fluid is important for the poisoning investigation. In this study, an online large‐volume sample stacking (LVSS) with polarity switching in capillary electrophoresis (CE) was applied for the sensitive determination of barbiturates. This technique involves injecting a large volume of sample into a capillary and removing the sample matrix plug out of the capillary by reversing the polarity. Quantitation limit obtained was 0.048, 0.057, 0.039, and 0.015 μg/mL for secobarbital, amobarbital, barbital, and phenobarbital (signal‐to‐noise ratio = 9). By using LVSS, the stacking was simply achieved at 171.7‐, 169.7‐, 202.7‐, and 169.1‐fold for the above four barbiturates. The relative standard deviation values of intraday and interday were <2.11% and 4.69%, respectively. Recoveries were ranged from 83.7 to 105.2%. Finally, the trace analysis method was applied to the analysis of real forensic specimens and has achieved satisfactory results.

Separation and determination of four ganoderic acids from dried fermentation mycelia powder of Ganoderma lucidum by capillary zone electrophoresis

Ganoderic acids (GAs) were bioactive secondary metabolites produced by a traditional mushroom Ganoderma lucidum. We describe a simple and efficient method for the separation and quantitative determination of four GAs, namely Ganoderic acid T (GA-T), Ganoderic acid Mk (GA-Mk), Ganoderic acid Me (GA-Me) and Ganoderic acid S (GA-S) from dried triterpene-enriched extracts of G. lucidum mycelia powder by capillary zone electrophoresis (CZE). Under the optimum conditions, the four GAs reached the baseline separation in 9 min with Glycyrrhetinic acid (GTA) as internal standard. The four GAs and internal standard (GTA) were detected at a wavelength 245 nm. All calibration curves showed good linearity (r(2)>0.9958) within test ranges. Limit of detection (LOD) and limit of quantification (LOQ) were less than 0.6 and 1.8 microg/mL, respectively. The relative standard deviation (R.S.D.) values of precision and recoveries were less than 5% and recoveries ranged from 91.4% to 103.6%. This was the first report on simultaneous determination of the four GAs and the results provided a firm basis for the trace analysis of GAs in dried fermentation mycelia powder of G. lucidum with high accuracy.