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Analytical and Bioanalytical Chemistry (v.385, #8)

Tips for effective poster presentations by Cynthia K. Larive; Ewa Bulska (pp. 1347-1349).

is Professor of Analytical Chemistry at the University of California Riverside. She has an active research program involving bioanalytical and environmental analytical applications of NMR Spectroscopy (for more information see http://www.chem.ucr.edu/faculty/larive/larive.html ). Professor Larive is also active in curricular reform and the promotion of undergraduate research. She is editor-in-chief and principal investigator of the Analytical Sciences Digital Library ( http://www.asdlib.org ), an Internet-based resource for instructors, students and practitioners of analytical chemistry. This digital library is a collection of peer-reviewed websites on topics including pedagogical approaches, analytical techniques, applications and classroom resources. is a Professor of Analytical Chemistry at the Warsaw University. Her research involves fundamental studies in analytical chemistry as well as the development and application of new methods for analytical measurements. She is very much involved in teaching and in the introduction of new pedagogical tools in the field of analytical chemistry. She is the Head of the Commission of Analytical Atomic Spectrometry of the Committee of Analytical Chemistry of the Polish Academy of Sciences. Her many awards include the Bunsen-Kirchoff Award 2004 for achievements in atomic spectroscopy.

Miniaturization—not just a matter of size... by Oliver Geschke (pp. 1350-1350).

Laser processing for bio-microfluidics applications (part I) by Chantal G. Khan Malek (pp. 1351-1361).

This paper reviews applications of laser-based techniques to the fabrication of microfluidic devices for biochips and addresses some of the challenges associated with the manufacture of these devices. Special emphasis is placed on the use of lasers for the rapid prototyping and production of biochips in particular for applications in which silicon is not the preferred material base. Part I of this review addresses applications and devices using UV lasers for laser ablation and surface treatment of microchannels, in particular in polymers.

Keywords: Laser; Micromachining; Bio-MEMS; μTAS; Microfluidics; Polymers

Laser processing for bio-microfluidics applications (part II) by Chantal G. Khan Malek (pp. 1362-1369).

This paper reviews applications of laser-based techniques to the fabrication of microfluidic devices for biochips and addresses some of the challenges associated with the manufacture of these devices. Special emphasis is placed on the use of lasers for the rapid prototyping and production of biochips, in particular for applications in which silicon is not the preferred material base. This review addresses applications and devices based on ablation using femtosecond lasers, infrared lasers as well as laser-induced micro-joining, and the laser-assisted generation of micro-replication tools, for subsequent replication of polymeric chips with a technique like laser LIGA.

Keywords: Laser; Micromachining; Bio-MEMS; μTAS; Microfluidics; Polymers

Towards biochips using microstructured optical fiber sensors by Lars Rindorf; Poul Erik Høiby; Jesper Bo Jensen; Lars Hagsholm Pedersen; Ole Bang; Oliver Geschke (pp. 1370-1375).

In this paper we present the first incorporation of a microstructured optical fiber (MOF) into biochip applications. A 16-mm-long piece of MOF is incorporated into an optic-fluidic coupler chip, which is fabricated in PMMA polymer using a CO₂ laser. The developed chip configuration allows the continuous control of liquid flow through the MOF and simultaneous optical characterization. While integrated in the chip, the MOF is functionalized towards the capture of a specific single-stranded DNA string by immobilizing a sensing layer on the microstructured internal surfaces of the fiber. The sensing layer contains the DNA string complementary to the target DNA sequence and thus operates through the highly selective DNA hybridization process. Optical detection of the captured DNA was carried out using the evanescent-wave-sensing principle. Owing to the small size of the chip, the presented technique allows for analysis of sample volumes down to 300 nL and the fabrication of miniaturized portable devices.

Keywords: Microstructured optical fiber; Biosensor; Miniaturization; Lab-on-a-chip; Packaging

Stabilization of two-phase octanol/water flows inside poly(dimethylsiloxane) microchannels using polymer coatings by H. J. van der Linden; L. C. Jellema; M. Holwerda; E. Verpoorte (pp. 1376-1383).

In this paper we present our first results on the realization of stable water/octanol, two-phase flows inside poly(dimethylsiloxane) (PDMS) microchannels. Native PDMS microchannels were coated with high molecular weight polymers to change the surface properties of the microchannels and thus stabilize the laminar flow profile. The polymers poly(2-hydroxyethyl methacrylate), poly(vinyl pyrrolidone), poly(ethylene oxide), poly(ethylene glycol), and poly(vinyl alcohol) were assessed for their quality as stabilization coatings after deposition from flowing and stationary solutions. Additionally, the influence of coating the microchannels homogeneously with a single kind of polymer or heterogeneously with two different polymers was investigated. From the experimental observations, it can be concluded that homogeneous polymer coatings with poly(2-hydroxyethyl methacrylate) and poly(vinyl pyrrolidone) led to the effective stabilization of laminar water/octanol flows. Furthermore, heterogeneous coatings led to two-phase flows which had a better-defined and more stable interface over long distances (i.e., 40-mm-long microchannels). Finally, the partitioning of fuchsin dye in the coated microchannels was demonstrated, establishing the feasibility of the use of the polymer-coated PDMS microchannels for determination of logP values in laminar octanol/water flows.

Keywords: Partition coefficient; Poly(dimethylsiloxane); Polymer coating; Two-phase flow; Microchannel

Bio-assay based on single molecule fluorescence detection in microfluidic channels by Christopher W. Hollars; Jana Puls; Olgica Bakajin; Brad Olsan; Chad E. Talley; Stephen M. Lane; Thomas Huser (pp. 1384-1388).

A rapid bioassay is described based on the detection of colocalized fluorescent DNA probes bound to DNA targets in a pressure-driven solution flowing through a planar microfluidic channel. By employing total internal reflection excitation of the fluorescent probes and illumination of almost the entire flow channel, single fluorescent molecules can be efficiently detected leading to the rapid analysis of nearly the entire solution flowed through the device. Cross-correlation between images obtained from two spectrally distinct probes is used to determine the target concentration and efficiently reduces the number of false positives. The rapid analysis of DNA targets in the low pM range in less than a minute is demonstrated.

Keywords: Biomedical assay; Colocalization; Image cross-correlation; Microfluidics; Single molecule fluorescence; TIRF

Fabrication of cell-containing hydrogel microstructures inside microfluidic devices that can be used as cell-based biosensors by Won-Gun Koh; Michael V. Pishko (pp. 1389-1397).

This paper describes microfluidic systems containing immobilized hydrogel-encapsulated mammalian cells that can be used as cell-based biosensors. Mammalian cells were encapsulated in three-dimensional poly(ethylene glycol)(PEG) hydrogel microstructures which were photolithographically polymerized in microfluidic devices and grown under static culture conditions. The encapsulated cells remained viable for a week and were able to carry out enzymatic reactions inside the microfluidic devices. Cytotoxicity assays proved that small molecular weight toxins such as sodium azide could easily diffuse into the hydrogel microstructures and kill the encapsulated cells, which resulted in decreased viability. Furthermore, heterogeneous hydrogel microstructures encapsulating two different phenotypes in discrete spatial locations were also successfully fabricated inside microchannels.

Keywords: Hydrogel microstructure; Cell-based biosensor; Microfluidic device; Poly(ethylene glycol)

Analysis of human breath with micro extraction techniques and continuous monitoring of carbon dioxide concentration by Wei Ma; Xinyu Liu; Janusz Pawliszyn (pp. 1398-1408).

The detection of volatile organic compounds (VOCs) in human breath can be useful for the clinical routine diagnosis of several diseases in a non-invasive manner. Traditional methods of breath analysis have some major technical problems and limitations. Membrane extraction with a sorbent interface (MESI), however, has many advantages over current methods, including good selectivity and sensitivity, and is well suited for breath analysis. The aim of this project was to develop a simple and reproducible sampling device and method based on the MESI system for breath analysis. The feasibility and validity of the MESI system was tested with real human breath samples. Internal standard calibration methods were used for the quantitative analysis of various breath samples. Calibration curves for some main components (target analytes such as acetone and pentane) were determined in the research. The optimized stripping-side and feeding-side gas velocities were determined. The use of breath CO₂ as an internal standard for the analysis of breath VOCs is an effective method to solve the difficulties associated with variations in the target analyte concentrations in a sample, which are attributed to mass losses and different breathing patterns of different subjects. In this study, the concentration of breath acetone was successfully expressed normalized to CO₂ as in the alveolar air. Breath acetone of healthy males and females profiled at different times of the day was plotted using the MESI system, and results were consistent with the literature. This technique can be used for monitoring breath acetone concentrations of diabetic patients and for applications with other biomarker monitoring.

Keywords: Membrane extraction with sorbent interface (MESI); CO₂ sensor; Internal standard; Breath analysis; Breath acetone

Removing coordinated metal ions from proteins: a fast and mild method in aqueous solution by Charlotte Carrer; Michael Stolz; Erwin Lewitzki; Claudia Rittmeyer; Bernd O. Kolbesen; Ernst Grell (pp. 1409-1413).

Thermodynamic and kinetic studies of metal binding to proteins require the investigation of metal-free proteins, which are often difficult to obtain. We have developed a very fast and mild method to eliminate metal ions from proteins by column chromatography using a commercially available Ni-NTA-type stationary phase. This material, initially designed for protein purification purposes in biotechnology, acts as a strong cation chelator when Ni²⁺ ions are removed. We have tested this new method with Ca-ATPase, an integral membrane protein exhibiting a strong affinity for Ca²⁺. By eluting the protein over the Ni²⁺-free NTA gel, we could remove 95% of the total Ca²⁺ and obtain an essentially Ca²⁺-free protein. This method is efficient with only a small amount of NTA gel, and we suggest that it can be applied in general for removal of metal ions from proteins. Moreover, as this procedure can be carried out under mild conditions, the chosen protein kept its enzymatic activity.

Keywords: Metal ion removal; Apoprotein; Ni-NTA chromatography; Ca-ATPase; TXRF; Fluorescent calcium indicator

A novel enhancement assay for immunochromatographic test strips using gold nanoparticles by Ryo Tanaka; Teruko Yuhi; Naoki Nagatani; Tatsuro Endo; Kagan Kerman; Yuzuru Takamura; Eiichi Tamiya (pp. 1414-1420).

The immunochromatographic assay is a well-known and convenient diagnostic system. In this report, the development of a novel enhancement assay for the test strips is described. Additionally, this highly sensitive immunochromatographic assay was applied to detect human chorionic gonadotropin hormone (HCG) as the model case. The primary antibody-conjugated gold nanoparticles were used as the enhancer of the standard method. The primary antibodies were immobilized within a defined detection zone (test line) on the diagnostic nitrocellulose membrane. The secondary antibodies were conjugated with colloidal gold nanoparticles. In combination with an effective sample pretreatment, the gold-conjugated antibodies and the primary antibodies formed a sandwich complex with the target protein. Within the test line, the sandwich complex was immobilized, and furthermore, concentrated by the enhancer resulting in a localized surface plasmon resonance (LSPR) phenomenon and a distinct red color on the test line. The intensity of color of the red test line (signal intensity), which correlated directly with the concentration of the target protein in the standard or spiked samples, was assessed visually and by computer image analysis using a three-determination analysis. Under optimum conditions, the limit of detection (LOD) for HCG assay was 1 pg/mL. When using human serum, 10 pg/mL of HCG could be detected. We have also spiked total prostate-specific antigen (TPSA) in female serum. The LOD for TPSA was determined as 0.2 ng/mL. With this method, the quantitative determination of the target protein could be completed in less than 15 min. Our novel immunochromatographic strips using the enhancing method based on LSPR of gold nanoparticles are useful as a rapid and simple screening method for the detection of important analytes for medical applications, environmental monitoring, food control, and biosecurity.

Keywords: Gold immunochromatographic assay; Colloidal gold nanoparticles; Human chorionic gonadotropin hormone; Total prostate-specific antigen

Characterisation of two novel cyclodextrinases using on-line microdialysis sampling with high-performance anion exchange chromatography by Carina Nilsson; Frida Nilsson; Pernilla Turner; Martin Sixtensson; Eva Nordberg Karlsson; Olle Holst; Arieh Cohen; Lo Gorton (pp. 1421-1429).

In this work, a real-time sampling/analytical method for on-line measurements of two newly discovered cyclomaltodextrinases (CDases) has been developed and evaluated. This novel methodology not only allows the final products to be investigated, but it also reveals enzyme-specific differences in the degradation pathways during the hydrolysis of different substrates, which is a great advantage in the important tasks of investigating the mechanisms of and classifying new hydrolases, and is an advantage that conventional techniques cannot offer. Two different enzymes, one CDase from Laceyella sacchari (LsCda13) and one from Anoxybacillus flavithermus (AfCda13), were investigated during the hydrolysis of α-, β- and γ-cyclodextrin, and the hydrolysis products were sampled via a microdialysis probe and injected on-line every 30 min into a high-performance anion exchange chromatography system equipped with a pulsed amperometric detector (HPAEC–PAD), where they were identified. The enzymes yielded the same end-products, maltose and glucose, in an approximate molar ratio of 2:1, but they exhibited distinctly different patterns of intermediate product formation before reaching the end-point. LsCda13 had a more random distribution of the intermediate products, whereas AfCda13 showed the distinct intermediate production of maltotriose, which in some cases accumulated.

Keywords: Enzyme hydrolysis; Carbohydrate analysis; Laceyella sacchari (LsCda13); Anoxybacillus flavithermus (AfCda13); Cyclodextrins (CD)

Measurement of hydrolysis kinetics of galactose-substituted fluorescein by β-galactosidase at the toluene–water interface by spinning microtube fluorometry by Yusuke Okamoto; Hideaki Monjushiro; Takao Fukumoto; Hitoshi Watarai (pp. 1430-1438).

A new analytical technique, spinning microtube fluorometry (SMF), was developed and applied to the study of interfacial hydrolysis of 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C₁₂FDG) by β-galactosidase (β-gal) in the toluene–water system. The nonfluorescent lactone form of C₁₂FDG in the toluene phase was converted at the interface to 5-dodecanoylaminofluorescein (C₁₂F), which was fluorescent in the aqueous phase as a dianion at pH 7.3, though some part of C₁₂F was extracted into the toluene phase as its nonfluorescent lactone form. The distribution ratios of C₁₂FDG and C₁₂F at pH 7.3 were determined as 1.4×10² and 1.97, respectively. The interfacial adsorption constants from the toluene phase to the interface at pH 7.3 were 4.8×10⁻⁴ and 1.7×10⁻² dm for C₁₂FDG and C₁₂F, respectively. The kinetic experiments with the SMF method concluded that the rate-determining step of the enzymatic hydrolysis at the interface and in the aqueous phase was the 1:1 reaction of C₁₂FDG and β-gal and that the hydrolysis reaction rate constant at the interface at pH 7.3 was 1.84×10³ M⁻¹s⁻¹, almost equal to that in the aqueous solution, 1.76×10³ M⁻¹s⁻¹. Finally, the SMF method revealed that the contribution of the interfacial reaction to the overall hydrolysis reaction rate of the toluene–water system was as high as 97%.

Keywords: Spinning microtube fluorometry; Interfacial enzymatic kinetics; Interfacial adsorption; β-Galactosidase; Liquid-liquid interface

UPLC-MS/MS determination of doxazosine in human plasma by Osama Y. Al-Dirbashi; Hassan Y. Aboul-Enein; Minnie Jacob; Khalid Al-Qahtani; Mohamed S. Rashed (pp. 1439-1443).

A sensitive, selective and rapid method for the analysis of doxazosine (DOX) in human plasma based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) is described. DOX and tamsulosin, used as an internal standard (IS), were extracted by liquid-liquid extraction, and the chromatography was performed on a C18 UPLC column packed with 1.7 μm particles. The total run time was 2 min. Detection was achieved by the multiple reaction monitoring of the following transitions: m/z 452→344 and m/z 409→228 for DOX and IS, respectively. Transitions of m/z 452→247 and m/z 409→271 were also collected for confirmation purposes. The calibration curve based on peak area ratio was linear up to at least 100 ng ml⁻¹, with a detection limit of 0.02 ng ml⁻¹ (a signal-to-noise ratio of 3). The method showed satisfactory reproducibility, and the short-term stability of the analyte was assessed. The method was successfully applied to the analysis of DOX in human plasma.

Keywords: Doxazosine; UPLC; UPLC-MS/MS

Determination of carbofuran, carbaryl and their main metabolites in plasma samples of agricultural populations using gas chromatography–tandem mass spectrometry by Syrago-Styliani E. Petropoulou; Anthony Tsarbopoulos; Panayotis A. Siskos (pp. 1444-1456).

A gas chromatography–tandem mass spectrometric (GC-MS/MS) method has been developed for the determination of carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate), carbaryl (1-naphthyl-N-methylcarbamate) and their main metabolites in human blood plasma. Optimization of the isolation of the compounds from plasma matrix included the precipitation, denaturation and digestion of plasma proteins. Derivatization was achieved by the use of trifluoroacetic acid anhydride and was optimized for temperature, time and volume of derivatization agent. In the proposed method, a mild precipitation technique was applied using β-mercaptoethanol and ascorbic acid in combination with solid-phase extraction technique using Oasis HLB (Hydrophobic Lipophilic Balance) cartridges for further clean up of samples. Carbamate linkage was not hydrolyzed to its phenol product, but both carbamate phenol and ketones were transformed into trifluoroacetyl derivatives in order to become volatile compounds and were determined using tandem mass spectrometry. The linearity of the method was shown for nine concentrations in the range of 0.50–250 ng mL⁻¹ in fortified plasma aliquots. Limits of detection (LODs) for all compounds ranged from 0.015–0.151 ng mL⁻¹. Inter-day and intra-day assays (RSD) for all compounds, at three concentration levels of 2.5, 25 and 100 ng mL⁻¹ (n=3) in fortified plasma samples were less than 18%. Accuracy (%E _r) was calculated at three concentration levels, 8, 80 and 160 ng mL⁻¹ (n=3), and ranged from −12.0 to 15.0%. Matrix effect was evaluated so mean recoveries were calculated for all compounds and ranged from 81–107%. Specificity for the use of this method to biological monitoring studies was achieved including four main metabolites of CF, 1-naphthol and 2-naphthol from the naphthalene metabolism pathways, and both the parent compound of carbofuran and carbaryl. The proposed method was applied to plasma samples of pesticide users.

Keywords: N-methylcarbamates; Tandem mass spectrometry; Biological monitoring; Biomarkers of exposure; Human plasma samples

Preparation of a novel composite particles and its application in the fluorescent detection of proteins by Hong-Qi Chen; Lun Wang; Yan Liu; Wei-Li Wu; A-Ni Liang; Xiao-Lei Zhang (pp. 1457-1461).

A new fluorescence method for the detection of proteins with novel composite nanoparticles (CdS/PPA) has been developed. The composite nanoparticles have been prepared through an in-situ polymerization method under ultrasonic irradiation. The surface of the composite nanoparticles was covered with functional groups (-COOH). These groups may play a major role in the improving the water solubility and biocompatibility of the nanoparticles. The composite particles is combined with proteins in NaAc-HCl buffer solution (pH=1.99), which can result in strong fluorescence, and the response is linearly proportional to the concentration of proteins. In λem/λex=650 nm/365 nm place (the stoke’ shift is 285 nm), its fluorescent strength reaches the maximum. Under the optimum conditions, the linear range is 0.10–20.0 μg·ml⁻¹ with the detection limit of 41 ng·ml⁻¹ for HSA, and 0.10–15.0 μg·ml⁻¹ with the detection limit of 35 ng·ml⁻¹ for Human γ-IgG . The method has been applied to the determination of the total protein in human serum samples collected from the hospital and the results are satisfactory.

Keywords: Synthesis; Composite nanoparticles; Fluorescence assay; Protein; Detection

Cholinesterase inhibition based determination of pancuronium bromide in biological samples by Vesna P. Stankov-Jovanović; Snežana D. Nikolić-Mandić; Ljuba M. Mandić; Violeta D. Mitić (pp. 1462-1469).

Pancuronium bromide (PCBr) inhibition effect on enzyme cholinesterase from pooled human serum (Che, EC 3.1.1.8 acylcholine acylhydrolase) was used for development of a spectrophotometric kinetic method for PCBr determination in human serum and urine. Optimal conditions for the basic and inhibitor reactions were established: pH=7.7 and substrate concentration c(benzoylcholine chloride)=1.33 mmol/L. Kinetic parameters were also determined: Michaelis-Menten’s constant K_M=0.40 mmol/L, maximal reaction rate V_max=52.2 μmol/L min, inhibition constant K_i=0,56 μmol/L and IC₅₀=1.31 μmol/L. Linear dependence between the reaction rate and inhibitor concentration exists in PCBr concentration range 8.20–68.25 nmol/L, which corresponds to the real sample concentrations from 0.328 to 2.730 μmol/L. The method detection and quantification limits were 2.01 nmol/L and 6.67 nmol/L, respectively. Precision of the method was tested for three pancuronium concentrations (10.70, 29.35 and 51.25 nmol/L). Relative standard deviation (RSD) was in the range 0.15–7.45%. Accuracy was examined by standard addition method. Influence of the substances usually present in serum and urine on the reaction rate was tested. The developed method was applied for PCBr content determination in serum model samples, urine model samples and in urine taken during surgery. The method has good sensitivity, accuracy, precision and it is suitable for clinical practice.

Keywords: Pancuronium bromide; Cholinesterase inhibition; Benzoylcholine; Spectrophotometric kinetic determination

Direct electrochemistry and electrochemical catalysis of immobilized hemoglobin in an ethanol–water mixture by Hui-Hong Liu; Yong-Qing Wan; Guo-Lin Zou (pp. 1470-1476).

Hemoglobin (Hb) was immobilized on a glassy carbon electrode (GCE) surface by konjac glucomannan (KGM). KGM hydrogel films on GCE have relatively high stabilities in aqueous–ethanol mixtures. The entrapped hemoglobin undergoes fast direct electron transfer reactions in aqueous–organic solvent mixtures. The peak current is bigger, the peak-to-peak separation smaller and the formal potential observed in the cyclic voltammogram is more negative for Hb–KGM/GCE in ethanol–PBS compared to Hb–KGM/GCE in PBS. The electrochemical properties of the Hb in aqueous–organic solution are almost unchanged from with those observed for the purely aqueous solution, suggesting that water pools in the KGM hydrogel play an important role in preventing changes in conformation and making proteins unreactive with polar organic solvents. The immobilized Hb was able to catalyze the reduction of nitric oxide, peroxides (hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, 2-butanone peroxide), and the dehalogenation of haloethanes (hexachloroethane, pentachloroethane, tetrachloroethane, etc.). The stability and reproducibility of the modified electrode meant that it could be used to determine these substances.

Keywords: Hemoglobin; Electrochemistry; Enzymatic activity; Peroxides; Chloroethanes

Ion-selective electrode for measuring low Ca²⁺ concentrations in the presence of high K⁺, Na⁺ and Mg²⁺ background by Iwona Bedlechowicz-Śliwakowska; Peter Lingenfelter; Tomasz Sokalski; Andrzej Lewenstam; Magdalena Maj-Żurawska (pp. 1477-1482).

In this work, ion-selective electrodes for calcium ion were investigated. Two ionophores were used in the membranes: ETH 1001 and ETH 129. An internal filling solution buffered for primary ion was used that allowed the lower detection limit to be decreased down to 10^−8.8 M. Theoretical and experimental electrode characteristics pertaining to both primary and interfering ions are discussed. Better behavior was obtained with the electrode prepared with ETH 129 in the membrane. This electrode would be the most likely candidate for obtaining a low Ca²⁺ detection limit in measurements performed with high K⁺, Na⁺, Mg²⁺ background, which is found inside the cells of living organisms, for example. The potentiometric response of the electrode in solutions containing main and interfering ions is in good agreement with simulated curves obtained using the Nernst–Planck–Poisson (NPP) model.

Keywords: Calcium ion-selective electrode; Selectivity; Detection limit

Usefulness of π...π aromatic interactions in the selective separation and analysis of imidazolium and pyridinium ionic liquid cations by P. Stepnowski; J. Nichthauser; W. Mrozik; B. Buszewski (pp. 1483-1491).

Current research indicates that replacing organic solvents with room-temperature ionic liquids may lead to remarkable improvements in well-known processes. Ionic liquids have already been utilized as alternative solvents in organic synthesis and catalysis, and also in electrochemistry and in separation sciences. Their wide applicability will soon result in their production on an industrial scale. Therefore, analytical methods applicable to various matrices for product control and environmental monitoring will be very much in demand. In this study, the usefulness of π...π interactions between alkylimidazolium and pyridinium cations and the aromatic π...π active moiety of the stationary phase was investigated for the selective separation and analysis of some ionic liquids. With phenyl-bonded phases, very good separations of the cations were achieved. Special attention was paid to the short-chain hydrophilic entities, known to be poorly separated on conventional reversed-phase columns. Further, the nature of the interactions occurring in the system under study was investigated by varying the content of methanol/acetonitrile used as organic modifiers in the mobile phases. The analytical method developed here is simple and reproducible, and its quantitative analytical performance was excellent. The paper also discusses the applicability of the method for monitoring degraded cations of 1-butyl-3-methylimidazolium salts obtained in the Fenton process.

Keywords: Ionic liquids; Analysis; π...π interactions; Liquid chromatography; Separation; Cations

Sensitivity improvement in capillary electrophoresis using organo-aqueous separation buffers and thermal lens detection by Svetlana N. Bendrysheva; Mikhail A. Proskurnin; Ute Pyell; Werner Faubel (pp. 1492-1503).

It is shown that organo-aqueous separation buffers show much promise when used in capillary electrophoresis separations with photothermal (thermal lens) detection systems. Acetonitrile–water and methanol–water mixtures were selected, as conventionally used in capillary electrophoresis. It is shown that, despite more sophisticated experimental conditions (significant heat outflow from the capillary body) and peak detection, the theoretical ratio of the thermal lens signal for a binary mixture to the thermal lens signal for an aqueous solution (or the corresponding ratio obtained experimentally under bulk batch conditions) can be used to predict the sensitivity of thermal lens detection in capillary electrophoresis. The limits of detection for 2-, 3-, and 4-nitrophenols selected as model compounds in 70% v/v acetonitrile separation buffers are 1×10⁻⁶ M, 1×10⁻⁶ M and 3×10⁻⁷ M, respectively, and are therefore decreased by a factor of six compared to thermal lens detection in aqueous separation buffers. The overall increase in the thermal lens detection sensitivity in a 100% ACN buffer is a factor of 13.

Keywords: Photothermal spectroscopy; Thermal lens detection; Capillary electrophoresis; Organo-aqueous solutions; Sensitivity; Media comparison

New separation method for organic and inorganic selenium compounds based on anion exchange chromatography followed by inductively coupled plasma mass spectrometry by L. Ayouni; F. Barbier; J. L. Imbert; J. Y. Gauvrit; P. Lantéri; M. F. Grenier-Loustalot (pp. 1504-1512).

We describe a new method for separating the organic and inorganic selenocompounds methaneseleninic acid, selenite, selenate, methylselenocysteine, selenocystine as well as both selenomethionine and its oxidized form. The separation is performed on a Hamilton PRP-X100 column. According to the literature, the oxidized form of selenomethionine—which is easily formed—is eluted close to the dead volume when this column is used. The choice of parahydroxybenzoic acid as mobile phase enabled us to elute all of these species after this oxidized form, resulting in better identification and quantification. The factors determining separation (eluent concentration, pH, gradient) were optimized via an experimental design. Application of the method to yeast and commercial tablets showed that the principal Se compound present was selenomethionine, which was also present in its oxidized form.

Keywords: Selenium; Speciation; Anion exchange chromatography; Experimental design

Preparative HPLC separation of bambuterol enantiomers and stereoselective inhibition of human cholinesterases by Ivana Gazić; Anita Bosak; Goran Šinko; Vladimir Vinković; Zrinka Kovarik (pp. 1513-1519).

We separated and characterized the enantiomers of bambuterol (5-[-(tert-butylamino)-1-hydroxyethyl]-m-phenylene-bis(dimethylcarbamate) hydrochloride), which is used in racemic form as a prodrug of terbutaline, a β₂-adrenoceptor agonist. The enantioseparation was attempted on several chiral HPLC columns, and the most effective separation was achieved on the amylose-based Chiralpak AD column. Since in vivo conversion of bambuterol into terbutaline involves hydrolysis by butyrylcholinesterase (EC 3.1.1.8), we studied the reaction of enantiomers with eight human BChE variants. Both enantiomers inhibited all studied BChE variants; however, the rate of inhibition with the (R)-enantiomer was about five times faster than with the (S)-enantiomer. (R)-bambuterol inhibition rate constants for homozygous usual (UU), fluoride-resistant (FF) or atypical (AA) variant ranged from 6.4 to 0.11 min^-1μM^-1. The inhibition rates for heterozygotes were between the respective constants for the corresponding homozygotes.

Keywords: Cholinesterase; Inhibition; Bambuterol; Enantiomers; Preparative HPLC

Sensitive determination of thiamethoxam, imidacloprid and acetamiprid in environmental water samples with solid-phase extraction packed with multiwalled carbon nanotubes prior to high-performance liquid chromatography by Qingxiang Zhou; Yujie Ding; Junping Xiao (pp. 1520-1525).

This paper describes a novel method for sensitive determination of thiamethoxam, imidacloprid and acetamiprid based on solid-phase extraction with multiwalled carbon nanotubes as the packed materials. Factors that maybe influence the enrichment efficiency, such as sample flow rate, sample pH, and sample volume, were investigated in detail. Under the optimized conditions, the detection limits of thiamethoxam, imidacloprid and acetamiprid were 6.1, 5.4 and 6.7 ng L⁻¹, respectively. The experimental results indicated that there was good linearity (R ²>0.9993) over the range of 0.08~100 ng mL⁻¹ and good reproducibility with the relative standard deviations over the range of 0.7~1.1% (n=6). The proposed method has been applied to the analysis of real-world water samples, and satisfactory achievements were obtained. The average spiked recoveries were in the range of 87.5~109.8%. All the results indicated that the proposed method could be used for the simultaneous determination of the three pesticides in environmental water samples at trace levels.

Keywords: Multiwalled carbon nanotubes; Solid-phase extraction; Thiamethoxam; Imidacloprid; Acetamiprid

Analysis of acrylamide in coffee and cocoa by isotope dilution liquid chromatography–tandem mass spectrometry by Patricia C. Aguas; Matthew J. Fitzhenry; Georgina Giannikopoulos; Peter Varelis (pp. 1526-1531).

An accurate and precise method for the quantification of acrylamide using stable isotope dilution liquid chromatography–tandem mass spectrometry was developed and used to measure acrylamide in coffee and cocoa samples. The sample preparation involved extraction of the analyte and its internal standard, ¹³C₃-acrylamide, into water and subsequent defatting of the aqueous extract with dichloromethane. An aliquot of the resulting aqueous extract was then azeotropically dried under reduced pressure and subsequently purified using an aminopropyl-bonded silica cartridge. The purified extracts were then chromatographed on a 5-μm 2.1×150 mm Hypercarb column, the effluent of which was monitored for the analyte and its internal standard using positive-ion APCI-selected reaction monitoring. The intra-laboratory reproducibility of the method, expressed as a relative coefficient of variation (%, n=5), was determined at four levels of concentration (12.3, 42.3, 139.3 and 464.8 μg kg⁻¹) and was found to vary between 0.6–2.5%. The accuracy of the method was assessed using a reference sample of coffee. The average result obtained using our method differed from the assigned value of the reference material by less than 1%. An analysis of a cocoa sample revealed that the method is capable of precisely estimating acrylamide in challenging matrices down to a level of at least 12.3 μg kg⁻¹.

Keywords: Acrylamide; Tandem mass spectrometry; Isotope dilution; Coffee; Cocoa; Atmospheric pressure chemical ionisation

FT-midIR determination of fatty acid profiles, including trans fatty acids, in bakery products after focused microwave-assisted Soxhlet extraction by J. Ruiz-Jiménez; F. Priego-Capote; M. D. Luque de Castro (pp. 1532-1537).

A study of the feasibility of Fourier transform medium infrared spectroscopy (FT-midIR) for analytical determination of fatty acid profiles, including trans fatty acids, is presented. The training and validation sets—75% (102 samples) and 25% (36 samples) of the samples once the spectral outliers have been removed—to develop FT-midIR general equations, were built with samples from 140 commercial and home-made bakery products. The concentration of the analytes in the samples used for this study is within the typical range found in these kinds of products. Both sets were independent; thus, the validation set was only used for testing the equations. The criterion used for the selection of the validation set was samples with the highest number of neighbours and the most separation between them (H<0.6). Partial least squares regression and cross validation were used for multivariate calibration. The FT-midIR method does not require post-extraction manipulation and gives information about the fatty acid profile in two min. The 14:0, 16:0, 18:0, 18:1 and 18:2 fatty acids can be determined with excellent precision and other fatty acids with good precision according to the Shenk criteria, R ²≥0.90, SEP=1–1.5 SEL and R ²=0.70-0.89, SEP=2–3 SEL, respectively. The results obtained with the proposed method were compared with those provided by the conventional method based on GC-MS. At 95% significance level, the differences between the values obtained for the different fatty acids were within the experimental error.

Keywords: Fatty acids profiles; Trans fatty acids; Multivariate calibration; FT-midIR

Development of a sequential injection anodic stripping voltammetry (SI-ASV) method for determination of Cd(II), Pb(II) and Cu(II) in wastewater samples from coatings industry by Allan C. Vieira dos Santos; Jorge C. Masini (pp. 1538-1544).

This paper describes the development and validation of a sequential injection (SI) anodic stripping voltammetry (ASV) method using the hanging mercury drop electrode for accumulation of the heavy metal cations Cu(II), Pb(II) and Cd(II). The method was applied to wastewater samples after proper acid digestion in open vessels to eliminate matrix effects. For a deposition time of 90 s at the flow rate of 10 μl s⁻¹, the detection limits of the method were 0.06, 0.09 and 0.16 μmol L⁻¹ for Cd, Pb and Cu, respectively. Under these conditions the linear dynamic range was between 0.20 and 9.0 μmol L⁻¹ and the sampling frequency was 30 analyses per hour. The relative standard deviation of the method was 3%(n=7) at the concentration level of 2.0 μmol L⁻¹. The accuracy of the method was evaluated by spiking the samples with known amounts of the metal cations, and by comparison with an independent analytical technique, the inductively coupled plasma atomic emission spectroscopy (ICP-AES). Average recoveries were around of 84%, and the results showed no evidence of systematic errors in comparison to the ICP-AES.

Keywords: Sequential injection analysis; Wastewater; Heavy metals; Coatings industry

Evaluation of extraction approaches linked to ELISA and HPLC for analyses of microcystin-LR, -RR and -YR in freshwater sediments with different organic material contents by Pavel Babica; Jiří Kohoutek; Luděk Bláha; Ondřej Adamovský; Blahoslav Maršálek (pp. 1545-1551).

The efficiencies of conventional extraction techniques and analytical methods (HPLC–DAD and ELISA) were investigated for analyses of microcystins (MCs) in sediments. Our results showed several limitations. First, the extraction efficiency strongly depends on the extraction solvent, and extraction with 5% acetic acid in 0.2% trifluoroacetic acid (TFA)–methanol was confirmed as being the most appropriate for three different sediments (recovery: 33.1–44.9% of total MCs according to HPLC analyses). Second, the recovery of MCs was affected by the type of sediment but did not clearly correlate with the content of organic carbon. These results suggest that the sorption of MCs onto inorganic materials such as clay minerals is probably a more important process than interactions of the MCs with organic sediment matter. Third, the structure of the MCs is another crucial factor that affects the sorption of MCs and their recovery from sediments. Hydrophilic MC-RR gave much lower recoveries (20.0–38.8%) than MC-YR (44.1–59.5%) or MC-LR (55.3–77.8%) from three different types of spiked sediments. Recovery results analysed with HPLC–DAD correlated well with ELISA analyses. Further, extraction with 5% acetic acid in 0.2% TFA–methanol was used for analyses of MCs in 34 natural sediment samples collected from Brno reservoir (Czech Republic) from April to October 2005. Concentrations of MCs in sediments ranged from 0.003 to 0.380 μg/g sediment d.m. (ELISA results) or 0.016–0.474 μg/g d.m. (HPLC results). These values are equivalent to 0.63–96.47 μg/L of sediment (ELISA) or 4.67–108.68 μg/L (HPLC), respectively. Concentrations of sediment MCs showed both temporal and spatial variability, with the highest MC contents observed in the spring (April and May) and the lowest concentrations in July and August. Our results demonstrate the suitability of the methods described here for studying the occurrence, fate and ecological role of MCs in the aquatic environment.

Keywords: Microcystin; Sediment; ELISA; HPLC–DAD

H-point standard addition method applied to solid-state stripping voltammetry: quantitation of lead and tin in archaeological glazes by A. Doménech-Carbó; M. T. Doménech-Carbó; J. V. Gimeno-Adelantado; F. Bosch-Reig (pp. 1552-1561).

A solid-state electrochemical application of the H-point standard addition method to the quantification of two depositable metals A and B, which produce strongly overlapped stripping peaks, is described. The method is based on the mechanical transference of mixtures of the solid sample plus a selected compound, of a reference depositable metal R, and of known amounts of a reference material containing A or B, to paraffin-impregnated graphite electrodes. After a reductive deposition step, voltammograms recorded for those modified electrodes immersed into a suitable electrolyte produce stripping peaks for the oxidation of all of the metals deposited. Measurement of the currents at selected potentials in overlapping peaks corresponding to the stripping of A and B permits the quantitation of these metals in the solid sample, while avoiding matrix effects. The method was applied to the simultaneous determination of Pb and Sn in archaeological glazes using PbCO₃ and SnO₂ as standards and ZnO as a reference material.

Keywords: H-point standard addition; Stripping voltammetry; Lead; Tin; Archaeological glazes

Determination of arsenic in diesel, gasoline and naphtha by graphite furnace atomic absorption spectrometry using microemulsion medium for sample stabilization by Geisamanda Pedrini Brandão; Reinaldo Calixto de Campos; Aderval Severino Luna; Eustáquio Vinicius Ribeiro de Castro; Honério Coutinho de Jesus (pp. 1562-1569).

A procedure for the determination of As in diesel, gasoline and naphtha at μg L⁻¹ levels by GFAAS is proposed. Sample stabilization was achieved by the formation of three component solutions prepared by mixing appropriate volumes of the samples propan-1-ol and nitric acid aqueous solution. This mixture resulted in a one-phase medium, which was indefinitely stable. No changes in the analyte signals were observed over several days in spiked samples, proving long-term stabilization ability. The use of conventional (Pd) and permanent (Ir) modification was investigated and the former was preferred. Central composite design multivariate optimization defined the optimum microemulsion composition as well as the temperature program. In this way, calibration using aqueous analytical solutions was possible, since the same sensitivity was observed in the investigated microemulsion media and in 0.2% v/v HNO₃. Coefficients of correlation larger than 0.999 and an As characteristic mass of 22 pg were observed. Recoveries (n=4) obtained from spiked samples were 98±4, 99±3 and 103±5%, and the limits of detection in the original samples were 1.8, 1.2 and 1.5 μg L⁻¹ for diesel, gasoline and naphtha, respectively. Validation was performed by the analysis of a set of commercial samples by independent comparative procedures. No significant difference (Student’s t-test, p<0.05) was observed between comparative and proposed procedure results. The total determination cycle lasted 4 min for diesel and 3 min for gasoline and naphtha, equivalent to a sample throughput of 7 h⁻¹ for diesel and 10 h⁻¹ for gasoline and naphtha.

Keywords: Arsenic; Petroleum derivates; Stabilization as microemulsion; Graphite furnace atomic absorption spectrometry (GFAAS)

Determining sulfamonomethoxine and its acetyl/hydroxyl metabolites in chicken plasma under organic solvent-free conditions by Naoto Furusawa (pp. 1570-1574).

A quantitative technique is described for a sample preparation followed by high performance liquid chromatography method for the simultaneous determination of sulfamonomethoxine and its metabolites, N ⁴-acetyl SMM and 2,6-dihydroxy SMM, in chicken plasma. The average recoveries, analytical total time, and limits of quantitation were ≥80% (relative standard deviations (SD) ≤6%), <30 min sample-1 (12 samples in 2 h), and ≤0.09 μg ml⁻¹, respectively. The procedure, performed under 100% aqueous conditions, uses no organic solvents and toxic reagents at all and is, therefore, harmless to the environment and humans.

Keywords: Sulfamonomethoxine; N ⁴-acetyl sulfamonomethoxine; 2,6-dihydroxy sulfamonomethoxine; Organic solvent-free analysis

Rapid analysis of native neomycin components on a portable capillary electrophoresis system with potential gradient detection by LingLing Yuan; HongPing Wei; HuaTao Feng; Sam F. Y. Li (pp. 1575-1579).

A simple method based on capillary electrophoresis with potential gradient detection was developed to separate and detect neomycin components within 4 min without a derivatization step. Satisfactory separation and good repeatability were obtained using a separation buffer composed of 1 mM ammonium citrate (pH 3.5). The linearity of the method ranged from 10 to 1000 ppm with a limit of detection for neomycin B of about 7 ppm. After a simple dilution and filtering pretreatment step, neomycin components in three real samples were successfully analyzed without any major interference. Due to its simplicity and reliability, this method could provide an excellent alternative to the assays currently listed in U.S. and European Pharmacopoeia. The experiments were performed on a portable capillary electrophoresis system and, hence, the method can be readily applied to field analysis and point-of-care testing. Figure Photo of portable CE-P2-PGD system

Keywords: Aminoglycoside antibiotics; Capillary electrophoresis; Neomycin; Potential gradient detection

Development of restricted-access media supports and their application to the direct analysis of biological fluid samples via high-performance liquid chromatography by N. M. Cassiano; V. V. Lima; R. V. Oliveira; A. C. de Pietro; Q. B. Cass (pp. 1580-1580).

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Analytical and Bioanalytical Chemistry (v.385, #8)