Dual-opposite multi-walled carbon nanotube modified carbon fiber microelectrode for microfluidic chip-capillary electrophoresis determination of methyl parathion metabolites in human urine.

Methyl parathion (MP) is a highly toxic organophosphate and its exposure may lead to substantial adverse effects to human health. The existence of 4-nitrophenol (4-NP) in the form of free phenol, glucuronide (4-NP-G) or as a sulfate ester (4-NP-S) can be used as biomarkers to assess the duration and extent of MP exposure. In this work, a MC-CE device incorporating post-CE amperometric detection using multi-walled carbon nanotubes (MWNTs) modified carbon fiber microelectrode (CFME) was fabricated and assessed for simultaneous determination of 4-NP, 4-NP-G, and 4-NP-S in human urine. The detection sensitivity and stability was greatly enhanced by the modification of MWNTs. The capability of the MC-CE device with dual MWNTs modified CFME for detecting impurity was assessed and reliability established by high recoveries from 95 to 97% for spiked MP biomarkers. The method developed is shown to provide a simple, sensitive, and reliable means for monitoring 4-NP, 4-NP-G, and 4-NP-S in human urine.

Determination of taurine in human tear fluid by capillary electrophoresis with indirect amperometric detection based on electrogenerated bromine.

A new method for the determination of taurine was developed based on indirect amperometric detection after capillary electrophoresis. A serial dual-electrode detector comprising an on column Pt film electrode (upstream electrode) and an end column Pt microdisk electrode (downstream electrode) was utilized to conduct the indirect amperometric detection. Bromide is oxidized to bromine at upstream electrode and reduced back to bromide at downstream electrode. Since taurine can react with bromine quantitatively and rapidly, its concentration can therefore be determined by the decrease of the current for bromine reduction at the downstream electrode. Principal experimental parameters governing the analytical performance were investigated and optimized. Under the optimal conditions, taurine can be baseline separated from interfering amino acids and the detection limit of 0.18 µM was obtained with a linear correlation coefficient of 0.999 over the concentration range of 0.5-60 µM. The developed method has been successfully applied in the determination of taurine in human tear fluid. The taurine level obtained was in good agreement with previous reports and recoveries for taurine spiked ranged from 92-95% with relative standard deviations within 4.6%, demonstrating the reliability of the developed method in the determination of taurine in human tear fluid.

A serial dual-electrode detector based on electrogenerated bromine for capillary electrophoresis.

A new serial dual-electrode detector for CE has been designed and fabricated for postcolumn reaction detection based on electrogenerated bromine. A coaxial postcolumn reactor was employed to introduce bromide reagent and facilitate the fabrication of upstream generation electrode by simply sputtering Pt film onto the outer surface of the separation capillary. Bromide introduced could be efficiently converted to bromine at this Pt film electrode and subsequently detected by the downstream Pt microdisk detection electrode. Analytes that react with bromine could be determined by the decrease of bromine reduction current at the downstream electrode resulting from the reaction between analytes and bromine. The effects of serial dual-electrode detector working conditions including electrode potentials, bromide flow rate, and bromide concentration on analytical performance were investigated using glutathione (GSH) and glutathione disulfide (GSSG) as test analytes. Under the optimal conditions, detection limits down to 0.16 µM for GSH and 0.14 µM for GSSG (S/N = 3) as well as linear working ranges of two orders of magnitude for GSH and GSSG were achieved. Furthermore, the separation efficiency obtained by our dual-electrode detector design was greatly improved compared with previous reported design. The developed method has been successfully applied to determine the GSH and GSSG impurity in commercial GSH supplement.

Assessment of adulteration of soybean proteins in dairy products by 2D microchip-CE device.

To determine the adulteration of soybean proteins in dairy product, a microchip-CE device was developed to isolate selected fraction of soybean and milk proteins in pI range from 5.5 ∼ 7.0 by 1D IEF, followed by ITP/CZE in the embedded capillary for preconcentration, separation and UV detection at 280 nm. Compared to IEF-CZE without ITP preconcentration, the enhancement factor (EF) in detection of soybean proteins was 20 times. Adulteration of 0.1% soybean protein in total dairy proteins can be detected in less than 10 min.

Microfluidic chip capillary electrophoresis coupled with electrochemiluminescence for enantioseparation of racemic drugs using central composite design optimization.

Optimization based on central composite design (CCD) for enantioseparation of anisodamine (AN), atenolol (AT), and metoprolol (ME) in human urine was developed using a microfluidic chip-CE device. Coupling the flexible and wide working range of microfluidic chip-CE device to CCD for chiral separation of AN, AT, and ME in human urine, a total of 15 experiments is needed for the optimization procedure as compared to 75 experiments using the normal one variable at a time optimization. The optimum conditions obtained are found to be more robust as shown by the curvature effects of the interaction factors. The developed microfluidic chip-CE-ECL system with adjustable dilution ratios has been validated by satisfactory recoveries (89.5-99% for six enanotiomers) in urine sample analysis. The working range (0.3-600 µM), repeatability (3.1-4.9% RSD for peak height and 4.0-5.2% RSD for peak area), and detection limit (0.3-0.6 µM) of the method developed are found to meet the requirements for bedside monitoring of AN, AT, and ME in patients under critical conditions. In summary, the hyphenation of CCD with the microfluidic chip-CE device is shown to offer a rapid means for optimizing the working conditions on simultaneous separation of three racemic drugs using the microfluidic chip-CE device developed.

Microfluidic chip-capillary electrophoresis with dynamic multi-segment standard addition for rapidly identifying nephrolithiasis markers in urine.

A microchip-CE device was fabricated for bed-side monitoring of nephrolithiasis biomarkers in urine by incorporating on-chip continuous passive mixing and standard addition to reduce sample matrix interference, increase sample throughput and eliminate accessories for active mixing. Under optimized conditions with buffer containing 20 mM borate and 0.5 mM CTAB at pH 10.3, sample and standards injected electrokinetically at -350 V for 10 s for online mixing in a Y-merging flow microchannel prior to CE separation and UV detection at 210 nm, both inhibitors (citrate, CA) and promoters (oxalate, OA and uric acid, UA) for nephrolithiasis can be separated and determined in human urine in a single run completed within 10 min after a simple 50-fold sample dilution and filtering. Satisfactory working ranges from 0.13-40, 0.25-40 and 0.025-40 mM, LOD 2.6, 6.1 and 0.7 µM, repeatability (%RSD, n=5) for migration time 1.40, 1.43, 0.47 and peak area 4.46, 6.10, 1.98, respectively, for CA, OA and UA are obtained for urine samples. The use of on-chip standard addition is shown to improve repeatability of the migration time, assist the identification of nephrolithiasis markers from difficult samples with noisy baseline and enlarge the working range for nephrolithiasis marker determination. The device developed can be used for both routine and emergency monitoring to deliver results on demand for bedside monitoring and public health protection. It provides an early detection of nephrolithiasis to enable timely treatments, ease anxiety of parents for neonates consuming suspected contaminated food, and quick results for patients in a critical condition.

2-D t-ITP/CZE determination of clinical urinary proteins using a microfluidic-chip capillary electrophoresis device.

To replace the time-consuming sample pretreatment procedure, a microfluidic chip-CE device incorporating on-chip sample desalting/preconcentration with transient isotachophoresis (ITP)/capillary zone electrophoresis (CZE) was fabricated to perform sequential on-chip sample pretreatment and CE determination of four urinary proteins in clinical samples. On-chip sample desalting, clean-up and analyte preconcentration enable removing interfering sample matrix prior to transferring analytes to separation capillary for transient ITP/CZE determination. Four important urinary proteins transferrin, ß2-microglobulin, human serum albumin (HSA) and immunoglobulin G (IgG), investigated were shown to achieve quantitation limits sufficiently high to meet medical requirements, sensitivity enhancement up to 40-fold and detection limits down to 0.3, 0.05, 0.6, 0.5 mg/L, respectively. Compared to the stacking effect, the use of a large sample size was found to be the major factor for sensitivity enhancement. The method reliability is established by close to 100% recoveries and statistical agreement of results from the method developed with currently used clinical radio-immunoassay method for all four proteins investigated. Moreover, an assay time of less than 10 min is needed in the method developed as compared to 7 h for the radio-immunoassay method.

Development of CE-dual opposite carbon-fiber micro-disk electrode detection for peak purity assessment of polyphenols in red wine.

A new dual opposite carbon-fiber micro-disk electrode detector was fabricated and tested for hyphenation with CE in the polyphenol determination. Under optimized conditions, CE-dual opposite carbon-fiber micro-disk electrode was found able to baseline separate and determine five important polyphenols (trans-resveratrol, (+)-catechin, (-)-epicatechin, quercetin and gallic acid) in red wine within 16 min with low detection limit (0.031-0.21 mg/L) and satisfactory repeatability (2.0-3.3% RSD, n=5). The opposite dual electrode enables simultaneous determination of CE eluents for current ratio measured at +0.8 and +1.0 V versus Ag/AgCl for the peak purity assessment. The capability to identify the presence of co-migrating impurities in given polyphenol peaks was demonstrated in a mixed standard solution with overlapping (+)-catechin and (-)-epicatechin peaks and in commercial red wine with unknown impurities and confirming the reliability for polyphenol quantitation in red wine with matching migration time and current ratio.

Determination of free bilirubin and its binding capacity by HSA using a microfluidic chip-capillary electrophoresis device with a multi-segment circular-ferrofluid-driven micromixing injection.

A PMMA microfluidic chip-CE device with a multi-segment circular-ferrofluid-driven micromixing injector has been developed for the determination of free bilirubin and its binding capacity by HSA at equilibrium. The design of the device and its fabrication by a low cost CO(2) laser are discussed for intended applications. Under optimized conditions, the total binding capacity of HSA for bilirubin was determined as 16.3±1.4 mg/l00 mL human serum (n=3) and residual binding capacity for bilirubin 9.8 mg/100 mL (n=3) in normal infants. To assess risk of hyperbilirubinemia, free bilirubin and residual binding capacity by HSA provide a better indicator than total bilirubin, as neonates with impaired bilirubin binding capacity could be detected. In addition, residual binding capacity provides an advanced indicator to predict the onset of hyperbilirubinemia before the appearance of free bilirubin. HSA down to 94 nL is used in each titration and a full assay of four titrations takes up 376 nL HSA, sufficient for newborns with HSA in microliter range. The device has shown capable to provide adequate margin of protection to detect an early rising level of bilirubin and impaired binding capacity prior to the onset of jaundice condition.

Microfluidic chip-capillary electrophoresis for two orders extension of adjustable upper working range for profiling of inorganic and organic anions in urine.

To meet the need for onsite monitoring of urine anions, a microfluidic chip-capillary electrophoresis device was designed, fabricated and tested to extend the upper CE working range for an enhancement up to 500 fold (100 fold for sample dilution and 5 folds for CE injection) in order to analyze highly variable anionic metabolites in urine samples. Capillaries were embedded between two PMMA plates with laser-fabricated microchannel patterns to produce the microfluidic chip-capillary electrophoresis to perform standard/sample dilution and CE injection with adjustable dilution ratios. A circular ferrofluid valve was incorporated on-chip to perform cleanup and conditioning, mixing and dilution, injection and CE separation. Under optimized conditions, a complete assay for four samples can be achieved within an hour for 15 anions commonly found in urines. Satisfactory working ranges (0.005-500 mM) and low detection limits (0.5-6.5 µM based on S/N =2) are obtained with satisfactory repeatability (RSD, n=5) 0.52-0.87% and 4.1-6.5% for migration time and peak area, respectively. The working ranges with two orders adjustable upper extension are adequate to cover all analytes concentrations commonly found in human urine samples. The device fabricated shows sufficiently large experimentally verifiable enhancement factor to meet the application requirements. Its reliability was established by more than 94% recoveries of spiked standards and agreeable results from parallel method comparison with conventional ion chromatography method. The extension of the upper CE working range enables flexible onsite dilution on demand, a quick turn-around of results, and a low-cost device suitable for bedside monitoring of patients under critical conditions for metabolic disorders.

Multidimensional microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula.

Functional proteins have been found in infant milk formula as supplements, added by an increasing number of manufacturers. Their supplementations are expected to be controlled and monitored. Here, we describe a microchip-integrated CE method for the determination of these low levels of functional proteins in a protein-rich sample matrix. On-chip isoelectric focusing (IEF) is used to separate high-abundance proteins from low-abundance proteins instead of using some complicated time-consuming protein purification process. After that, transient isotachophoresis hyphenated capillary zone electrophoresis (t-ITP-CZE) can preconcentrate, separate, and analyze transferred functional proteins in the embedded capillary under UV detection.

Correction of retention time shifts for chromatographic fingerprints of herbal medicines.

In this study, the combination of chemometric resolution and cubic spline data interpolation was investigated as a method to correct the retention time shifts for chromatographic fingerprints of herbal medicines obtained by high-performance liquid chromatography-diode array detection (HPLC-DAD). With the help of the resolution approaches in chemometrics, it was easy to identify the purity of chromatographic peak clusters and then resolve the two-dimensional response matrix into chromatograms and spectra of pure chemical components so as to select multiple mark compounds involved in chromatographic fingerprints. With these mark components determined, the retention time shifts of chromatographic fingerprints might be then corrected effectively. After this correction, the cubic spline interpolation technique was then used to reconstruct new chromatographic fingerprints. The results in this work showed that, the purity identification of the chromatographic peak clusters together with the resolution of overlapping peaks into pure chromatograms and spectra by means of chemometric approaches could provide the sufficient chromatographic and spectral information for selecting multiple mark compounds to correct the retention time shifts. The cubic spline data interpolation technique was user-friendly to the reconstruction of new chromatographic fingerprints with correction. The successful application to the simulated and real chromatographic fingerprints of two Cortex cinnamomi, fifty Rhizoma chuanxiong, ten Radix angelicae and seventeen Herba menthae samples from different sources demonstrated the reliability and applicability of the approach investigated in this work. Pattern recognition based on principal component analysis for identifying inhomogenity in chromatographic fingerprints from real herbal medicines could further interpret it.

Determination of volatile components in ginger using gas chromatography-mass spectrometry with resolution improved by data processing techniques.

Ginger is widely used as either a food product or an herbal medicine in the world. In this paper, a method was developed for determining volatile components in essential oils from both dried and fresh ginger by use of gas chromatography-mass spectrometry (GC-MS) and chemometric approaches. With the resolution improvement by chemometric methods upon two-dimensional data from GC-MS, the drifting baseline can be corrected. In addition, the peak purity can be assessed and the number of chemical components and their stepwise elution in the peak clusters can be identified. The peak clusters investigated are then resolved into pure chromatograms and related mass spectra for each of the components involved. Finally, with the pure chromatograms and related mass spectra obtained, the chemical components can be qualitatively identified based on the similarity searches in the MS databases and the chromatographic retention times. Quantitative determination can be conducted using the overall volume integration approach. The results showed that 140 and 136 components were separated and that 74 and 75 of them were tentatively identified, which accounted for about 62.82 and 47.11% of the total relative content for dried and fresh ginger, respectively. In comparison with the chromatographic fingerprints of essential oils from dried and fresh ginger, 60 of the volatile components determined match with each other. The study demonstrated that the use of chemometric resolution based on two-dimensional data can mathematically enhance the separation ability of GC-MS and assist qualitative and quantitative determination of chemical components separated from complicated practical systems such as foods, herbal medicines, and environmental samples.

Analysis of volatile components from Cortex cinnamomi with hyphenated chromatography and chemometric resolution.

In this paper, the combination of hyphenated chromatography and chemometric resolution was investigated as a method to qualitatively and quantitatively determine volatile components in Cortex cinnamomi from four main producing areas. With the help of chemometric resolution approaches, whether the chromatographic elution of chemical components is featured by "first-in-first-out" or embedded peaks could be determined. Upon this useful information obtained, the matrix data generated by hyphenated chromatography could be uniquely resolved into pure chromatogram and spectrum of each chemical component involved followed by qualitative and quantitative analysis. The results obtained in this work showed that, 94, 88, 93 and 89 volatile components were separated and 63, 60, 60 and 58 of them qualitatively and quantitatively determined representing about 93.39, 93.62, 92.03 and 92.59% of the total relative content, respectively. The combination of hyphenated chromatography with chemometric resolution could greatly enhance the chromatographic separation and spectral qualitatively determination ability so as to qualitatively and quantitatively detect many more volatile components and improve the analysis accuracy.

Simultaneous determination of gaseous and particulate carbonyls in air by coupling micellar electrokinetic capillary chromatography with molecular imprinting solid-phase extraction.

A novel method coupling molecular imprinting solid-phase extraction (MISPE) and micellar electrokinetic capillary chromatography (MEKC) was developed to enable the hourly determination of low level of ambient carbonyls, and study their partition between gaseous phase and particulate phase. With 2,4-dinitroaniline (DNAN) as dummy imprinting template, the unreacted 2,4-Dinitrophenylhydrazine (DNPH) in sampling solution could be removed effectively using MISPE, and an average recovery of 97±5.3% (n=5) for the carbonyl-DNPH derivatives was achieved. Owing to the high enrichment due to sample clean-up, and the improvement of MEKC separation efficiency, many low abundant carbonyls could be detected by hourly in the field study.

Multi-dimension microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula.

To improve resolution of important minor proteins and eliminate time-consuming precipitation of major protein with associated analyte co-precipitation risk, a multi-dimension strategy is adopted in the 2D microchip-CE device to isolate major proteins on-chip, enrich minor proteins in capillary before their separation in CE for UV quantitation. A standard fluorescent protein mixture containing FITC-BSA, myoglobin and cytochrome as specific pI markers has prepared to demonstrate capability of the device to fractionate minor proteins by IEF. The results using a standard protein mixture with profile resembling infant milk formula show a complete isolation of high abundance proteins by a 2-min 1D IEF run. The subsequent t-ITP/CZE run by on-chip high voltage switching delivers a high stacking ratio, realizing 60 folds enrichment of isolated protein fractions. All five important functional proteins (LF, IgG, α-LA, ß-LgA and ß-LgB) known to fortify infant milk formula are isolated and determined using two consecutive t-ITP-CZE runs within a 18-min total assay time, a significant saving compared to several hours conventional pretreatment. For a 100g infant milk formula sample, working ranges of 20-8000mg, repeatability 3.8-5.3% and detection limits 2.3-10mg have been achieved to meet government regulations. Method reliability is established by 100% recoveries and agreeable results within expected ranges and labeled values. The capability of the device for field operation, rapid assay with quick results, label-free universal detection, simple operation by aqueous dissolution before injection, and the demanding matching in 2D separation based on isolated fractions at specified pI ranges, closely matched migration time and baseline-resolved peak shape makes the device a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples with interfering constituents.

Microchip capillary electrophoresis for frontal analysis of free bilirubin and study of its interaction with human serum albumin.

To meet the need for bedside monitoring of free bilirubin for neonates under critical conditions, a microfluidic chip was fabricated and tested for its coupling with CE/frontal analysis (FA) to determine free bilirubin and study of its binding interaction with HSA, which regulated its concentration in plasma. The poly(methyl methacrylate) (PMMA) multichannel chip was fabricated by CO2 laser ablation and bonded with a fused-silica separation capillary for CE/FA separation with UV detection. The chip was designed to allow a complete assay of four electrophoretic runs using preconditioned channels to speed up the determination of free bilirubin and to deliver quick results for bedside monitoring. Under optimized conditions, the linear working range for free bilirubin was from 10 to 200 micromol with RSDs from 2.1 to 5.0% for n=3, and the LOD at 9 micromol for S/N=3. From a binding study between bilirubin and HSA under FA condition, the second binding constant for bilirubin-HSA was determined as 1.07x10(5) L/mol and the number of binding sites per HSA as 3.46. The results enabled the calculation of free bilirubin for jaundiced infants based on the clinically significant level of total bilirubin, producing a range of 118.3-119.4 micromol/L. The developed method is shown to meet the clinical requirement with additional margin of protection to detect the early rising level of free bilirubin prior to jaundice condition. The low-cost microchip CE/FA device is shown to produce quick results with high potential to deliver a suitable bed-side monitoring method for bilirubin management in neonates.

Determination of gaseous and particulate carbonyls in air by gradient-elution micellar electrokinetic capillary chromatography.

A new continuous-flow gradient-elution micellar electrokinetic capillary chromatography method is developed for the determination of airborne carbonyls after derivatization with 2,4-dinitrophenylhydrazine. A total of 16 carbonyls can be determined with detection limits ranging from 0.94 to 8.50 mg/L, working range from 4.72 to 346 mg/L, and repeatabilities (relative standard deviation, n=5) from 1.23 to 4.6% or 3.93 to 7.6% for migration time and peak area, respectively. Coupling with denuder-filter sampling, a preliminary survey has been conducted to determine gaseous and particulate carbonyls from air sampled at a roadside station. The method is shown to have sufficient sensitivity for 1-h sampling of ambient carbonyls with detection limits ranging from 0.045 to 1.2 microg/m3 and working range from 0.11 to 43.3 microg/m3 at a flow rate of 10 Lpm. The method requires minimal modification of commercially available capillary electrophoresis equipment and can differentiate gaseous and particulate carbonyls to provide essential information and objective data for adopting effective measures to combat the discharge of carbonyl compounds to the atmosphere.

Separation and determination of metal cations in milk and dairy products by CE.

To prevent casein adsorption and improve between-run repeatability, a CE procedure is developed for cation analysis in milk using a modified imidazole/alpha-hydroxyisobutyric acid (HIBA) BGE system to operate at pH 6 to match the pH of milk and elevate imidazole concentration to enhance its buffer action. The procedure is shown to produce a fast, economic and efficient method for cation separation in milk with only simple dilution. Upon direct hydrodynamic injection of diluted milk sample at 8 cm for 30 s in an uncoated column with a BGE consisting of 10 mM imidazole, 10 mM HIBA and 10% methanol at pH 6.0 under +18 kV, baseline separation was achieved for K(+), Na(+), Ca(2+), Mg(2+), Mn(2+), Cd(2+), Co(2+), Ni(2+)and Zn(2+). Agreeable results at 95% confidence level were obtained using CE and inductively coupled plasma-atomic emission spectrometry (ICP-AES) for milk samples after protein removal. Baseline-resolved peaks for essential minerals were obtained for fresh and non-refrigerated reconstituted milks. Long-term stability was demonstrated by repeated determinations without rinsing and improvement in repeatability was shown by rinsing with 60 mM SDS in BGE. Information on metal speciation useful for nutritional assessment was obtained from CE to complement the ICP methods.

Variable selection for discriminating herbal medicines with chromatographic fingerprints.

When discriminating herbal medicines with pattern recognition based on chromatographic fingerprints, typically, the majority of variables/data points contain no discrimination information. In this paper, chemometric approaches concerning forward selection and key set factor analysis using principal component analysis (PCA), unweighted and weighted methods based on the inner- and outer-variances, Fisher coefficient from the between- and within-class variations were investigated to extract representative variables. The number of variables retained was determined based on the cumulative variance percent of principal components, the ratio of observations to variables and the factor indicative function (IND). In order to assess the methods for variable selection and criteria levels to determine the number of variables retained, the original and reduced datasets were compared with Procrustes analysis and a weighted measure of similarity. Moreover, the tri-variate plots of the first three PCA scores were used to visually examine the reduced datasets in low dimensional space. Herbal samples were finally discriminated by use of Bayes discrimination analysis with the reduced subsets. The case study for 79 herbal samples showed that, the methods of forward selection associating the variables with the loadings closest to 0 and key set factor analysis were preferable to determine the representative variables. Procrustes analysis and the weighted measure were not indicative to extract representative variables. High matching between the original and reduced datasets did not suggest high prediction accuracy. Visually examining the PC1-PC2-PC3 scores projection plots with the reduced subsets, not all the herb samples could be separated due to the complexity of chromatographic fingerprints.