Capillary electrophoresis

This work reports the validation of a simple CZE method to be used in quality control of recombinant human glycosylated interleukin-7 (rhIL-7) batches produced in Chinese Hamster Ovary (CHO) cells. The separation buffer was a 25mM sodium borate at pH 10 containing 12mM diaminobutane (DAB) used as a dynamic coating agent of the capillary. This method allowed the separation of seven peaks ranging from low to high sialylated glycoforms. An extensive study on conditioning methods of the capillary has been conducted to yield repeatable results. Excellent RSD of EOF mobility (less than 0.6%) was obtained when conditioning included capillary equilibration under virtual analyses and storage in 0.1M NaOH overnight. Method specificity has been demonstrated to be able to discriminate different rhIL-7 glycoforms produced in CHO from formulation matrix. Linearity was demonstrated between 0.5 and 4mg/mL. LOQ was 0.5mg/mL. Repeatability (RSD<1.4 and 3.3% for t(m) and A%, respectively), intermed...

The use of microfluidic glass chips for continuous single-cell lysis and assay of internal-Galactosidase (-Gal) content is described. Cells were transported single file toward a Y-shaped mixing junction at which lytic agents were introduced by suction. Flow velocities of 100 and 40 m/s were used under protein denaturing [35 mM sodium dodecylsulfate (SDS)] and nondenaturing (0.1% Triton X-100) conditions, respectively. Complete and reproducible lysis of individual cells on-chip occurred within 30 s using Triton X-100 and 2 s when using SDS. Optimal concentrations of lysis and enzyme substrate reagents were determined using microtitre plate and chip-based procedures. Fluorescence peaks, due to the enzymatic product fluorescein mono-D-galactopyranoside (FMG), were detected downstream of the mixing and cell lysis point for the reaction of-Gal with 200 M of the fluorogenic substrate fluorescein-diD -galactopyranoside (FDG). FMG fluorescence was observed from cells preincubated with FDG off-chip then subsequently lysed on-chip with SDS. Unincubated cells were mixed on-chip with both FDG and Triton X-100, each individual cell generating FMG fluorescence downstream of the mixing point detected within 2 min of mixing. In contrast, viable cells incubated with FDG required 1 h or more in order to generate significant signal in a flow cytometer.

We investigated the application of two technologies for the fabrication of rectangular microchannels with reasonable path lengths for UV detection in UV transparent materials. The first approach uses inductively coupled plasma (ICP)-reactive ion etching (RIE) to fabricate channels in fused silica wafers, using a 4-mm-thick nickel layer as protective mask. The effects of the process parameters on the etched channel profile and surface quality were studied directly using electron scanning microscopy, and indirectly through capillary electrophoresis experiments. In the second approach, narrow channels were easily realized in poly(dimethylsiloxane) (PDMS) using replica molding and dry-etched silicon masters. UV absorbance detection of tryptophan was possible vertically through these PDMS channels, using pre-aligned optical fibers to guide light to the channel and collect and bring the transmitted light to the detector. 

Miniaturized, portable instrumentation has been gaining popularity in all areas of analytical chemistry. Capillary electrophoresis (CE), due to its main strengths of high separation efficiency, relatively short analysis time and low consumption of chemicals, is a particularly suitable technique for use in portable analytical instrumentation.

The present study optimised the accelerated solvent extraction (ASE) conditions (Dionex ASEÒ 200, USA) to maximise the antioxidant capacity of the extracts from three spices of Lamiaceae family; rosemary, oregano and marjoram. Optimised conditions with regard to extraction temperature (66-129 °C) and solvent concentration (32-88% methanol) were identified using response surface methodology (RSM). For all three spices results showed that 129 °C was the optimum temperature in order to obtain extracts with high antioxidant activity. Optimal methanol concentrations with respect to the antioxidant activity of rosemary and marjoram extracts were 56% and 57% respectively. Oregano showed a different response to the effect of methanol concentration and was optimally extracted at 33%. The antioxidant activity yields of the optimal ASE extracts were significantly (p < 0.05) higher than solid/liquid extracts. The predicted models were highly significant (p < 0.05) for both total phenol (TP) and ferric reducing antioxidant property (FRAP) values in all the spices with high regression coefficients (R2) ranging from 0.952 to 0.999.

A new, moderately hydrophobic, single-isomer charged cyclodextrin, the sodium salt of heptakis(2,3-diacetyl-6sulfato)-cyclodextrin, has been synthesized and used to separate a variety of neutral, weak acid, strong base, weak base, and zwitterionic racemic enantiomers in low-pH and high-pH background electrolytes. Separation selectivity for the netural analytes rapidly decreases as the concentration of heptakis(2,3-diacetyl-6-sulfato)-cyclodextrin increases. For charged analytes, selectivity can increase, decrease, or pass a maximum, depending on the numeric values of the respective complexation constants and ionic mobilities. In addition to separation selectivity, the extent of peak resolution that can be realized strongly depends on the magnitude of the dimensionless electroosmotic flow. Heptakis(2,3-diacetyl-6-sulfato)-cyclodextrin proved to be a broadly applicable chiral resolving agent.

On-line conversion and determination of aspirin using a flow injection-capillary electrophoresis system A novel, rapid, and simple capillary electrophoresis (CE) method has been developed for the determination of aspirin by on-line treatment with alkali. Such on-line reaction with 0.20 mol/L NaOH solution for 2.5 min at 458C automatically and reproducibly converts aspirin into its hydrolysis product salicylic acid. Analysis is carried out in less than 12 min after conversion of aspirin in a flow injection (FI) system coupled to a CE instrument via a split-flow interface cell, and a sampling frequency of 9 h -1 is achievable. The on-line conversion method has been used to determine aspirin in four commercial pharmaceutical preparations, and the results are satisfactory.

Cisplatin is for a long time in clinical use as efficient antitumor drug. The success in cisplatin-based chemotherapy, however, strongly depends on how careful the drug's dosages are monitored in order to reduce severe side-effects and overcome cellular resistance. The use of micellar electrokinetic chromatography with direct UV detection is described for the determination of intact cisplatin in human serum. The main product of drug's hydrolytic metabolism, cis-diammineaquachloroplatinum(II), was quantified using capillary zone electrophoresis in combination with indirect UV detection and on-line transient isotachophoresis preconcentration. The detection limits of platinum species studied were about 2-3 mol l −1 that allows the proposed methods to be applied for quantification of the administered levels of cisplatin as well as drug's active metabolite. Changes in the speciation of cisplatin occurring after intravenous administration can also be monitored using these simple and convenient CE techniques.

This report describes separation and detection of chlorophenoxy acid herbicides spiked in drinking water by the technique combining solid-phase extraction, fieldamplified sample stacking, capillary electrophoresis, and potential gradient detection. The herbicide solution (400 mL) was concentrated to 0.1 mL by the solid-phase extraction procedure. The buffer containing 3 mM ammonia and 0.3 mM hydroxypropyl-bcyclodextrin was adjusted to pH 9.0 with ammonia. The sample solution was injected into the capillary to 30% of the whole length, and 29 kV and 9 kV were employed for field-amplified sample stacking and separation, respectively. The herbicides were baseline separated and the detection limits with the above combined techniques were in the range of 1-4610 22 ng/mL.

Methods to discriminate plant oils facilitate the detection of either deliberate or accidental adulteration. To this direction, the variability in length among plant species of the chloroplast trnL intron was exploited for the authentication of edible and cosmetic plant oils, with an extra emphasis on olive oil. The methodology was based on the combinatorial use of a PCR assay with a capillary electrophoresis system such as the lab-on-a-chip technology. Application of the assay on DNA extracted from different oil producing plant species, including olive oil and sesame oil, indicated the ability of the trnL intron to be used as an analytical target. Furthermore, this assay could be used for the detection of adulteration of olive oil with various other plant oils, with the exception of avocado and sesame oil.

The potential of capillaries noncovalently coated with charged polymers for the metabolic analysis of body fluids by CE is illustrated. Firstly, the usefulness of a coating consisting of a triple layer of polybrene-dextran sulfate-polybrene for the fast analysis of organic acids is described. The CE system allowed direct injections of CSF, plasma and urine samples, yielding good separation efficiencies. RSDs for migration times and peak areas of organic acids in plasma were <3% and <5%, respectively. The usefulness of the system is illustrated by the profiling of organic acids in plasma and urine samples. Secondly, a CE system comprising a bilayer coating of polybrene-poly(vinylsulfonate), which provides a considerable EOF at low pH is described. This system was combined with TOF-MS and used for the fast analysis of amino acids in cerebrospinal fluid (CSF) and urine with minimal sample pretreatment. RSDs for migration times and peak areas of amino acids in CSF and urine were <2% and <10%, respectively. The applicability of the system is demonstrated by the profiling of endogenous low-molecular weight metabolites in CSF from a healthy individual and a patient with complex regional pain syndrome.

Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding site of human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimental conditions (HEPES buffer 50 mM, pH 7.4 and 25 °C) by means of isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique is based on measurements of a different property of the biochemical system, and then the results allow a critical discussion about the suitability of each approach to estimate the drug-protein binding parameters. The strongest interaction step is properly evaluated by the three assayed approaches being the derived binding constants strongly consistent: from 4 × 104 to 7 × 104 for HSA and from 0.8 × 105 to 1.2 × 105 for BSA. Binding enthalpy variations also show consistent results: -5.4 and -5.6 Kcal/mol for HSA and -4.3 and -3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively...

We present a method to size DNA fragments from 50 to 800 bp on a DNA analyser using an alternative size standard (X-Rhodamine MapMarker 1000) run on a capillary electrophoresis system (Applied Biosystem's 3730 DNA Analyser). This method avoids several limitations imposed by manufacturer-developed size standards, instruments and software and offers a more robust approach to conducting DNA size fragment analyses.

In this work, a methodological approach is reported, aimed at assessing the electrochemical response of some model gluco-oligosaccharides (dextrans). Such strategy is based on the complementary use of both anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD) and capillary zone electrophoresis coupled with UV detection (CZE–UV). Unlike HPAEC–PAD, CZE–UV required derivatization with a chromophoric dye (i.e., 8-aminonaphtalene-1,3,6-trisulphonic acid, ANTS) to enhance UV response and separation selectivity. From the comparison between chromophore response and PAD signal, the reliability of HPAEC–PAD for quantitative evaluation of dextran mixtures containing mainly oligomers with polymerization degree (DP) up to 18 could be proved, due to the fairly constant molar response. For higher DPs (up to 41), a maximum in the trend of the molar responses was observed followed by a steep decrease for DPs higher than about 30–35; indeed, an underestimation of weight-average...

To apply capillary electrophoresis for rapid screening for Y microdeletions A set of 25 specific sequence tagged sites that cover the azoospermia factor a, b, and c regions of the Y chromosome was amplified in 5 fluorescent multiplex sets each including 5 primer pairs. One of the primers of each pair was labeled with a fluorescent tag attached to the 5&#39;-end. After PCR amplification, analysis of the obtained PCR products was performed using capillary electrophoresis (ABI Prism 3100 Genetic Analyzer). The method was employed to determine Y microdeletions in azoospermic (n = 49) and severe oligozoospermic (n = 149) men. The number of PCR cycle (from 45 to 30) and the amount of DNA template (20-fold) used in fluorescent multiplex PCR were reduced because of the high sensitivity of capillary electrophoresis. Approximately 1000 multiplex PCR sets from 198 patients were analyzed simultaneously within 50 h. Y microdeletions were found in 3 out of the 198 azoospermic or severe oligozoosp...

Organic solvent modi®cation is investigated as a simple and effective way to decrease the conductivity of liquid samples containing anionic As species, making them suitable for ®eld ampli®ed sample stacking capillary electrophoresis (CE) measurements with direct UV detection. Experimental variables that in¯uence the procedure, as buffer pH, stacking and running potential, are described for methanol, selected as the optimum sample modi®er. The in¯uence of modifying the CE buffer with methanol on the resolution is also discussed. Results of stacking with and without removing the sample matrix from the capillary (with and without polarity switching after the stacking stage) are reported. The optimized method permits the determination of As(III) (arsenite), free As(V) (arsenate), and dimethylarsinate (DMA), in the range from 0.5 to 20 mg/l, with detection limits around 0.3 mg/l. For other As(V) compound of human origin, phenylarsonate (PAA), and p-aminobenzenearsonate (PABA), the power of detection is higher due to their higher extinction coef®cients. The linear range extends from 20 to 1000 mg/l, with a detection limit of 11 mg/l (PAA) and 16 mg/l (PABA). #

This paper proposes a novel method to fabricate multi-layers, embedded micro¯uidic structures by simply employing dosage-controlled UV exposure on thick SU-8 resist and anti-re¯ection coating on the bottom surface to prevent the re¯ection UV-light from inducing exposure. Experimental results show the top wall thickness of the embedded micro-channels can be well controlled in a resolution of 2 mm for the UV dosage from 120 to 190 mJ/cm 2. Stacked micro-channels have also been successfully realized and showed no interference on the bottom structures when the top structures are being exposed. Numerical simulation of the top wall thickness by UV exposure dosage control has also been conducted, and the comparison between the calculated and experimental results showed similarity in trend. This simple and inexpensive method can be applied to fabricate multi-layers of complex¯uidic systems for the applications of mTAS (MicroTotal Analysis System), inkjet printhead, capillary electrophoresis, and micro PCR (Polymerase Chain Reaction).

In this work, a new partial filling affinity capillary electrophoresis (PF-ACE) method has been developed and applied to investigation of non-covalent molecular interactions between double stranded DNA oligonucleotide (Dickerson dodecamer) and classical DNA intercalator ligand-ethidiumbromide (EtBr) or oligophenylene derivatives-based potential new type of DNA ligands. Binding constants of DNA-ligand complexes were determined from the dependence of migration time changes of DNA oligomer (applied as analyte) on the length of ligand zones introduced beforehand as plugs of various lengths (0-75 mm with 12.5 mm step) in hydroxypropylcellulose coated fused silica capillary of 50/375 m I.D./O.D. and 400/300 mm total/effective length. PF-ACE experiments were performed in two background electrolytes, Tris-borate, pH 8.0, ionic strength 14.3 mM (BGE1), and sodium phosphate, pH 7.5, ionic strength 133 mM (BGE2). Binding constants of DNA-EtBr complex (ca 15 300 L/mol in the BGE1 and 4200 L/mol in the BGE2) were found to be significantly higher than those of DNA complexes with oligophenylene derivatives (ca 2200-3600 L/mol in the BGE1 and 1600-2300 L/mol in the BGE2).

An improved and efficient method for the determination of underivatized amino acids based on the use of CE coupled to evaporative light scattering detector (ELSD), involving carbon nanotubes, was successfully developed. Carboxyled single-walled carbon nanotubes were used for the first time to perform the clean-up of the analyzed samples, which were afterwards analyzed by CE-ELSD. White tea samples were used to demonstrate the usefulness of the CE-ELSD coupled methodology. A suitable interface, based on a triple tube design sprayer, was developed and successfully used for coupling both instruments. Parameters affecting the separation and determination, including the elimination of interferences, were studied and properly optimized. Under the optimized conditions good resolution was achieved for the separation of seven amino acids. The precision of the method, expressed as RSD, was found within the 3.5-5.3% range. The LOD obtained for the proposed method were in the 1.2-2.1 pg range and the LOQ, were in the 2.0-11.5 pg range, with injection pressure of 5 KPa for 20 s (15.3 nL). This method is simple, rapid, and selective compared with other conventional techniques.

The cytolytic seed protein enterolobin from seeds ofEnterolobium contortisiliquumwas purified by using FPLC on a Mono Q column giving a single peak in capillary electrophoresis. The complete amino acid sequence of the plant cytolysin was determined by an automated method, yielding a molecular mass of 54,806 Da. Databank searches and sequence alignment demonstrated a high degree of sequence identity and

The objective of the current study was to develop and validate a simple, precise and accurate isocratic stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) assay method for the determination of spironolactone and furosemide in solid pharmaceutical dosage forms. Isocratic RP-HPLC separation was achieved on an SGE 150 × 4.6 mm SS Wakosil II 5C8RS 5-μm column using a mobile phase of acetonitrile-ammonium acetate buffer (50:50, v/v) at a flow rate of 1.0 mL/min. The detection was carried out at 254 nm using a photodiode array detector. The drug was subject to oxidation, hydrolysis, photolysis and heat to apply stress conditions. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The method was found to be linear in the drug concentration range of 40-160 µg/mL with correlation coefficients of 0.9977 and 0.9953 for spironolactone and furosemide, respectively. The precision (relative standard deviation; RSD) among a six-sample preparation was 0.87% and 1.1% for spironolactone and furosemide, respectively. Repeatability and intermediate precision (RSD) among a six-sample preparation were 0.46% and 0.20% for spironolactone and furosemide, respectively. The accuracy (recovery) was between 98.05 and 100.17% and 99.07 and 100.58% for spironolactone and furosemide, respectively. Degradation products produced as a result of stress studies did not interfere with the detection of spironolactone and furosemide; therefore, the assay can be considered to be stability-indicating.

The purpose of this study was to develop an analytical method to quantify the relative activities of carboxylesterases (CESs) in biological samples. Taking the advantage of loperamide, a specific carboxylesterase 2 (CES2) inhibitor, and bis-p-nitrophenyl phosphate (BNPP), an irreversible CESs inhibitor, we propose for the first time a capillary electrophoresis (CE) method that enables detecting and distinguishing CES2 activity from other CESs in complex biological samples. The capillary electrophoresis method proved to be fast, simple, repeatable, and applicable to the measurement of the specific activity of CESs. The method was successfully applied to the evaluation of human cells overexpressing human carboxylesterase 2 (hCE-2) and to several mammalian sera, using extremely small amounts of samples in comparison with traditional spectrophotometric methods. The same rationale can be applied to establish methods for determining the activity of other isoenzymes, using the appropriate specific inhibitors.

Binding between an aptamer and its target is highly dependent on the conformation of the aptamer molecule, this latter seeming to be affected by a variety of cations. As only a few studies have reported on the interactions of monovalent or divalent cations with aptamers, we describe herein the use of ACE in its mobility shift format for investigating interactions between various monovalent (Na 1 , K 1 , Cs 1 ) or divalent (Mg 21 , Ca 21 , Ba 21 ) cations and a 30-mer lysozyme-binding aptamer. This study was performed in BGEs of different natures (phosphate and MOPS buffers) and ionic strengths. First, the effective charges of the aptamer in 30 mM ionic strength phosphate and MOPS (pH 7.0) were estimated to be 7.4 and 3.6, respectively. Then, corrections for ionic strength and counterion condensation effects were performed for all studies. The effective mobility shift was attributed not only to these effects, but also to a possible interaction with the buffer components (binary or ternary complexes) as well as possible conformational changes of the aptamer. Finally, apparent binding constants were calculated for divalent cations with mathematical linearization methods, and the influence of the nature of the BGE was evidenced. Electrophoresis 2010, 31, 546-555 Condition 1: no IS correction K d 5 1.870.9 a) K d 5 5.372.5 K d 5 3.170.2 K d 5 5.870.5 K d1 5 0.370.0 (0-1 mM Mg 21 ) K d1 5 1.070.0 (0-2 mM Mg 21 ) K d2 5 3.570.5 (2-30 mM Mg 21 ) K d2 5 12.172.8 (3-30 mM Mg 21 ) Condition 2: IS correction with z 5 z opt 5 3.6 in MOPS or 7.4 in phosphate K d 5 0.670.1 N/A K d 5 1.770.4 K d 5 3.270.2 K d1 5 0.270.0 (0-1 mM Mg 21 ) K d2 5 1.270.3 (2-30 mM Mg 21 )

he central nervous system (CNS) -the brain and spinal cord -and the peripheral nervous system (PNS) together regulate multiple body functions. The neurones communicate with other organs and cell types, as well as with nervous tissue, by neurotransmission. Neurotransmitters are generally released from the synapses into the synaptic cleft, the space between the preand post-synaptic membranes, in very small volumes and low concentrations. Most neurotransmitters fall into one of three chemical categories: amino acids, amines or peptides, and they can be excitatory or inhibitory in their role of action. The mammalian immune system comprises organized lymphoid tissue, circulating cells and soluble proteins, such as immunoglobulins, cytokines and complement factors, that control immune homeostasis in the whole body. Communication within the immune system is largely dependent on cytokines, many of which have been described along with their effects on various immunocompetent cells.

Capillary electrophoresis (CE) was applied as a fast method of siderophore separation. Siderophores are iron binding and regulating cell products, which facilitate iron transport into cells. A fast and efficient method of siderophore analysis is important for better understanding of the iron pathways in a sea environment or marine organisms. The best results of CE analysis were obtained using free zone CE in 25 mM phosphate buffer at basic pH using a constant voltage of 20 kV. Under these conditions it was possible to detect the presence of siderophores in seawater.

In 1997, low-level perchlorate contamination (&lt;50 ng mL -1 or parts per billion) was discovered in the western U.S. Since that time, it has been found in sites scattered around the nation. Although the Environmental Protection Agency has not established a regulation for perchlorate in drinking water, it has placed perchlorate on the contaminant candidate list (CCL) and the unregulated

Environmental microbiology studies commonly use terminal restriction fragment length polymorphism (T-RFLP) of 16S rRNA genes, for example, to analyze changes in community structure in relation to changing physicochemical and biological conditions over space and time. Although T-RFLP is most useful for comparing samples from different environments, a large number of samples makes effective analysis difficult using the Web-based tools that are currently available. To resolve this dilemma, we used a new approach for calculating data from multiple T-RFLP samples by estimating terminal fragment combinations, then applying a correlation analysis using two different fluorescent dyes generated simultaneously from all samples. This calculation was based on the expectation that the proportions of two terminal fragments from one full-length polymerase chain reaction fragment would be nearly the same in each analysis. Using this program, the oral microflora in 73 human saliva samples were analyzed, and 24 bacterial groups, with peak areas of at least 0.5% and correlation coefficients of 0.55 or greater, were identified from the T-RFs within 40 s.

A method based on solid-phase extraction (SPE) and capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) is described for the separation and determination of six cytokinin nucleotides in coconut water. The best CZE separation for the six cytokinin nucleotide standards was achieved using a 25 mM ammonium formate/formic acid buffer (pH 3.8) and 2% (v/v) methanol with an applied gradient separation voltage (25 kV for 32 min, and then a linear gradient to 30 kV in 5 min, finally 30 kV to the end of separation) in less than 60 min. MS/MS with multiple reaction monitoring (MRM) detection was carried out to obtain sufficient selectivity and sensitivity for the cytokinin nucleotides. The combined use of on-line sample stacking and CZE-MS/MS achieved limits of detection (LODs) in the range of 0.06-0.19 M for the six cytokinin nucleotides at a signal-to-noise ratio of 3. Furthermore, a novel dual-step SPE procedure was developed for the pre-concentration and purification of cytokinin nucleotides using Oasis HLB and Oasis MAX cartridges. The recoveries of the cytokinin nucleotides after the dual-step SPE were in the range of 44-71%. The combination of off-line SPE, on-line sample stacking and CZE-MS/MS approach was successfully applied to screen for endogenous cytokinin nucleotides present in coconut water sample. trans-Zeatin riboside-5 -monophosphate (ZMP) was detected and quantified in coconut water by CZE-MS/MS after SPE and on-line sample stacking.

We have found that the Haarhoff-Van der Linde (HVL) peak function provides excellent fitting to the shapes of CZE peaks. Initially designed for overloaded peaks in gas chromatography, this function describes a Gaussian peak when there is no peak distortion, and a triangular peak when there is no diffusional peak broadening. As such, it is ideal for CZE peaks distorted by electromigration dispersion (EMD). Fitting peaks with this function gives four parameters: three of them can be related to the Gaussian peak that would have been obtained in case of no EMD; the last one is a measure of the peak distortion. Using moving boundary theory, this peak distortion parameter may readily be expressed in terms of analyte and background electrolyte mobilities and concentrations, electric field, and sample injection length. The variance of an HVL peak is shown to be described by a universal function, and a master equation is presented. The region where EMD adds less than 10% to the Gaussian variance is shown to be very narrowly spread around the mobility matching condition. Under typical CZE operating conditions with an analyte at 1% of the BGE concentration, significant peak distortion is always present. Because the total peak variance is not an addition of the Gaussian and triangular contributions, the HVL model and the methodology introduced here should always be used to correctly combine variances.

A high-yield synthesis of O-allyl ␤-d-galactopyranoside was carried out by the use of Aspergillus oryzae ␤-galactosidase. The reaction was carried out employing p-nitrophenyl ␤-d-galactopyranoside as the donor and a large excess of allyl alcohol as the acceptor. The molar yield was 65.6%, corresponding to an improvement of 41.3% with respect to the best results previously reported with other systems, and of 80.2% with respect to the results obtained using the same enzyme.

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.

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Leather industries which promote hide stabilization by the conventional chrome-tanning process are a major source of pollution because of the resultant chromium-rich wastes. In this work, an extensive characterization of such a chromium-rich waste sludge is presented, regarding its chemical composition (XRF), crystalline phase contents (XRD), organic carbon content (TOC), thermal behavior by thermogravimetry (TG) and differential scanning calorimetry (DSC), as well as its stability under chemical attack (the concentration of important ions in the leachates being determined by capillary electrophoresis) and when submitted to temperatures as high as 1100 8C, in air. The material showed the tendency to produce some undesirable, and previously non-detected hexavalent chromium when exposed to high temperatures, but after washing off the soluble salts and the elimination of the organic matter by firing, the resultant material was succesfully tested as a ceramic pigment in a conventional glaze composition usually employed in the ceramic tile industry.

In this work, a high-resolution CGE method for quantification and purity determination of recombinant proteins was developed, involving a single-component inclusion bodies (IBs) solubilization solution. Different recombinant proteins expressed as IBs were used to show method capabilities, using recombinant interferon-1b as the model protein for method validation. Method linearity was verified in the range from 0.05 to 0.40 mg/mL and a determination coefficient (r 2) of 0.99 was obtained. The LOQs and LODs were 0.018 and 0.006 mg/mL, respectively. RSD for protein content repeatability test was 2.29%. In addition, RSD for protein purity repeatability test was 4.24%. Method accuracy was higher than 90%. Specificity was confirmed, as the method was able to separate recombinant interferon-1b monomer from other aggregates and impurities. Sample content and purity was demonstrated to be stable for up to 48 h. Overall, this method is suitable for the analysis of recombinant proteins in IBs according to the attributes established on the International Conference for Harmonization guidelines.

A sensitive method for the determination of berberine, a plant alkaloid, was developed by capillary electrophoresis coupled with laser induced fluorescence detection. With the addition of (2-hydroxypropyl)-␤-cyclodextrin (2-HP-␤-CD) to the separation electrolyte, the low fluorescence signal intensity of berberine in aqueous medium was enhanced considerably by 162 fold. The optimal background electrolyte was selected as 20 mM acetic acid, 35 mM 2-HP-␤-CD, and 20% methanol at pH 5. The limit of detection of the method for berberine was 15.7 ng/mL. The intra and interday repeatabilities of corrected peak areas as RDSs were 1.83% and 3.75%, respectively. The developed method was successfully applied for the determination of berberine contents in Chinese medicinal plants and herbal supplemental tablets.

This paper provides an overview on the current status of the analysis of biogenic amines by CE. The basic CE separation and detection strategies for the analysis of biogenic amines are briefly described. CZE and MEKC that provide highly efficient and reproducible analysis of biogenic amines are particularly surveyed. With respect to the detection of biogenic amines, we focus on LIF, UV-visible absorption, electrochemiluminescence, and MS. Derivatization strategies, indirect methods, and on-line concentration techniques such as field-amplified sample stacking, sweeping, and use of polymer solution are described. To show the practicality of CE, we highlight currently developed techniques for the determinations of biogenic amines in biological samples, including foods, beverages, cerebrospinal fluids, urine, and single cells.

The separation and detection of common mono-and disaccharides by capillary electrophoresis (CE) with contactless conductivity detection (CCD) is presented. At high values of pH, the sugars are converted to anionic species that can be separated by CE and indirectly detected by CCD. The main anionic species present in the running electrolytes are hydroxide and phosphate, which have greater mobility than the ionized sugars, and, thus, the indirect detection is possible. The method was applied to analysis of glucose, fructose, and sucrose in soft drinks, isotonic beverages, fruit juice, and sugarcane spirits. Galactose was used as internal standard in all cases. Plate numbers range from ca. 70 700 to 168 200 and the limits of detection from 13 to 31 mM.

Background: Glutathione is a ubiquitous thiol-containing tripeptide, which plays a central role in cell biology. It is implicated in the cellular defence against xenobiotics and naturally occurring deleterious compounds, such as free radicals and hydroperoxides. Glutathione status is a highly sensitive indicator of cell functionality and viability. Its levels in human tissues normally range from 0.1 to 10 mM, being most concentrated in liver (up to 10 mM) and in the spleen, kidney, lens, erythrocytes and leukocytes. In humans, GSH depletion is linked to a number of disease states including cancer, neurodegenerative and cardiovascular diseases. The present review proposes an analysis of the current knowledge about the methodologies for measuring glutathione in human biological samples and their feasibility as routine methods in clinical chemistry. Furthermore, it elucidates the fundamental role of glutathione in pathophysiological conditions and its implication in redox and detoxification process. Tests available: Several methods have been optimised in order to identify and quantify glutathione forms in human biological samples. They include spectrophotometric, fluorometric and bioluminometric assays, often applied to HPLC analysis. Recently, a liquid chromatography-mass spectrometry technique for glutathione determination has been developed that, however, suffers from the lack of total automation and the high cost of the equipment. Conclusion: Glutathione is a critical factor in protecting organisms against toxicity and disease. This review may turn useful for analysing the glutathione homeostasis, whose impairment represents an indicator of tissue oxidative status in human subjects. D

A novel approach for CE data analysis based on pattern recognition techniques in the wavelet domain is presented. Low-resolution, denoised electropherograms are obtained by applying several preprocessing algorithms including denoising, baseline correction, and detection of the region of interest in the wavelet domain. The resultant signals are mapped into character sequences using first derivative information and multilevel peak height quantization. Next, a local alignment algorithm is applied on the coded sequences for peak pattern recognition. We also propose 2-D and 3-D representations of the found patterns for fast visual evaluation of the variability of chemical substances concentration in the analyzed samples. The proposed approach is tested on the analysis of intracerebral microdialysate data obtained by CE and LIF detection, achieving a correct detection rate of about 85% with a processing time of less than 0.3 s per 25 000-point electropherogram. Using a local alignment algorithm on low-resolution denoised electropherograms might have a great impact on high-throughput CE since the proposed methodology will substitute automatic fast pattern recognition analysis for slow, human based time-consuming visual pattern recognition methods.