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Analytica Chimica Acta (v.584, #1)

Contents (pp. v-vi).

Editorial Board (pp. co1).

Calendar of forthcoming meetings (pp. n1).

An enzyme-linked immunosorbant assay using polyclonal antibodies against bacopaside I by Watoo Phrompittayarat; Waraporn Putalun; Hiroyuki Tanaka; Sakchai Wittaya-Areekul; Kanchalee Jetiyanon; Kornkanok Ingkaninan (pp. 1-6).

Bacopa monnieri (L.) Wettst. (Brahmi) is a medicinal plant used as a memory enhancer in Ayurvedic medicines. Its active components are triterpenoid glycosides namely pseudojujubogenin and jujubogenin glycosides. In order to analyze these saponin glycosides, an enzyme-linked immunosorbant assay (ELISA) was developed using polyclonal antibodies against bacopaside I, one of the pseudojujubogenin glycosides found in the plant. Bacopaside I was conjugated with a bovine albumin serum (BSA) to prepare an immunogen. The bacopaside I–BSA conjugate was immunized to a rabbit for producing polyclonal antibodies (PAbs). The results showed that the antibodies were raised specifically against pseudojujubogenin glycosides. An ELISA using anti-bacopaside I PAbs was performed in the range of 1.95–62.5ngmL⁻¹ of bacopaside I and the limit of detection was 0.1ngmL⁻¹. The method was validated and the applicability of the ELISA for analyzing saponin glycosides from Brahmi was demonstrated.

Keywords: Bacopaside I; Bacopa monnieri; Enzyme-linked immunosorbant assay (ELISA); Pseudojujubogenin glycosides; Polyclonal antibodies

Biofunctional organic nanocrystals for quantitative detection of pathogen deoxyribonucleic acid by Cangel Pui-yee Chan; Lawrence Chi-hung Tzang; King-keung Sin; Sheng-lin Ji; Kwan-yee Cheung; Tze-kin Tam; Michael Meng-su Yang; Reinhard Renneberg; Matthias Seydack (pp. 7-11).

Advances in nanotechnology have had significant impacts in the field of biodiagnostics. In this study, we describe the novel application of dissolvable, organic and biofunctional nanocrystals for the quantitative detection of a PCR product. Fluorescein diacetate (FDA), a fluorogenic precursor of fluorescein, was milled in a solution of a polymeric surfactant to create a stable, nanosized colloid with an interface for coupling streptavidin molecules. The application of these particulate labels for the quantitative detection of biotinylated human papillomavirus (HPV) DNA, amplified in a standard PCR procedure, was demonstrated. After the affinity reaction, the FDA molecules were dissolved and concomitantly converted into fluorescein. This approach resulted in a high selectivity, short incubation times and a sensitivity up to 147 times greater than obtained from state-of-the-art, directly fluorescent-labeled streptavidins. This innovative method offers rapid detection of small amounts of nucleic acids because less target material and thus fewer PCR cycles are required.

Keywords: Fluorescein diacetate; Fluorescein 5-isothiocyanate; Horseradish peroxidase; Human papillomavirus

A novel one cycle allele specific primer extension—Molecular beacon displacement method for DNA point mutation detection with improved specificity by Xiaomin Li; Yong Huang; Yuan Guan; Meiping Zhao; Yuanzong Li (pp. 12-18).

We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3′ (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the “wrong” genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3′ exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3′-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3′-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.

Keywords: Polymerase chain reaction; Molecular beacon; Point mutation; Genotyping

Interaction between quercetin–copper(II) complex and DNA with the use of the Neutral Red dye fluorophor probe by Yongnian Ni; Shan Du; Serge Kokot (pp. 19-27).

The interaction of quercetin–Cu(II) complex with calf thymus DNA was investigated with the use of Neutral Red (NR) dye as a spectral probe by the application of UV–vis spectrophotometry, cyclic voltammetry and synchronous fluorescence spectroscopy. The results showed that both quercetin–Cu(II) complex and the NR molecule can intercalate into the double helix of the DNA. The 2:1 quercetin:Cu(II) complex (estimated binding constant=2.85×10⁹) is stabilized by intercalation in the DNA (binding constant, K_{[quercetin–Cu(II)–DNA]}=(1.82±0.20)×10⁵M⁻¹), and displaces the NR dye from the NR–DNA complex in a competitive reaction. Cyclic voltammetry studies confirm the intercalation reaction and show that the ratio ( K_R/ K_O) of binding constants for the reduced and oxidized forms of the metal complex is 2.05. Furthermore, the alternative least squares (ALS) method was applied to resolve a complex two-way array of the absorption spectra data. This yielded the equilibrium concentration profiles of each component in the reaction (NR, NR–DNA and quercetin–Cu(II)) as well as the corresponding pure spectra. The extracted profiles showed that at equilibrium the [NR–DNA] and [NR] trends decreased and increased symmetrically, respectively, with approximately linear behaviour being observed below 10×10⁻⁶molL⁻¹ of the added quercetin–Cu²⁺ complex. Thereafter, these trends converged asymptotically. The free [quercetin–Cu(II)] trend-line at equilibrium was linear over the whole range of the complex added. It was possible to estimate the approximate value of the equilibrium constant of the exchange process (approximately 5×10⁻¹) involving the intercalation of the quercetin–Cu(II) complex. It was also found that about 35% of the bound complex was unaccounted by the intercalation reaction, presumably being stabilized at an alternative site.

Keywords: Quercetin–Cu(II) complex; DNA; Neutral Red dye fluorescence probe; Intercalation; Alternative least squares

The characterization of (±)- anti-benzo[ a]pyrene diolepoxide–DNA adducts and (±)- anti-dibenzo[ a,l]pyrene diolepoxide–DNA adducts in the same DNA sample using solid-matrix phosphorescence by Allison L. Thompson; Robert J. Hurtubise (pp. 28-36).

Solid-matrix phosphorescence (SMP) spectra and lifetimes were used to characterize the (±)- anti-benzo[ a]pyrene diolepoxide [(±)-anti-B[ a]PDE] and (±)- anti-dibenzo[ a,l]pyrene diolepoxide [(±)- anti-DB[ a,l]PDE] bonded to the same sample of DNA. SMP spectra and lifetimes were also acquired for two samples of DNA that had only (±)-anti-B[ a]PDE or (±)- anti-DB[ a,l]PDE bonded to the individual samples of DNA. A detailed comparison of the SMP properties was made among the three samples of DNA. The SMP excitation spectra for the (±)-anti-B[ a]PDE–DNA and the (±)- anti-DB[ a,l]PDE–DNA adducts were very similar. However, the SMP emission spectra of the two DNA adduct systems were very dissimilar with a major emission band for the (±)-anti-B[ a]PDE–DNA adducts appearing at 613nm and for the (±)- anti-DB[ a,l]PDE–DNA adducts a major band was at 558nm. It was possible to selectively use SMP emission wavelengths and obtain a SMP excitation of spectrum of the (±)- anti-DB[ a,l]PDE–DNA adducts in the dual adducted DNA sample without the (±)-anti-B[ a]PDE–DNA adducts emitting SMP. In addition, it was shown that the SMP emission spectrum of the dual adducted DNA sample could be used to detect both adduct systems in the modified DNA sample. It was demonstrated that the SMP lifetimes could be effectively employed to characterize the dual adducted DNA sample. For example, the SMP decay curve for the (±)- anti-DB[ a,l]PDE–DNA adducts could be acquired without any SMP emission from the (±)-anti-B[ a]PDE–DNA adducts. Also, ln(SMP intensity) versus time plots were very useful in characterizing the dual adducted DNA sample.

Keywords: Solid-matrix phosphorescence; Heavy-atom effect; Benzo[; a; ]pyrene diol epoxide–DNA adducts; Dibenzo[; a; ,l]pyrene diolepoxide–DNA adducts

Prediction of T-cell epitopes based on least squares support vector machines and amino acid properties by Shuyan Li; Xiaojun Yao; Huanxiang Liu; Jiazhong Li; Botao Fan (pp. 37-42).

T-lymphocyte (T-cell) is a very important component in human immune system. It possesses a receptor (TCR) that is specific for the foreign epitopes which are in a form of short peptides bound to the major histocompatibility complex (MHC). When T-cell receives the message about the peptides bound to MHC, it makes the immune system active and results in the disposal of the immunogen. The antigenic determinants recognized and bound by the T-cell receptor is known as T-cell epitope. The accurate prediction of T-cell epitopes is crucial for vaccine development and clinical immunology. For the first time we developed new models using least squares support vector machine (LSSVM) and amino acid properties for T-cell epitopes prediction. A dataset including 203 short peptides (167 non-epitopes and 36 epitopes) was used as the input dataset and it was randomly divided into a training set and a test set. The models based on LSSVM and amino acid properties were evaluated using leave-one-out cross-validation method and the predictive ability of the test set, and obtained the results of 0.9875 and 0.9734 under the ROC curves, respectively. This result is more satisfactory than that were reported before. Especially, the accuracy of true positive gets a marked enhancement.

Keywords: T-cell epitopes; Classification; Least squares support vector machines; ROC curve

An untargeted metabolomics approach to contaminant analysis: Pinpointing potential unknown compounds by A. Lommen; G. van der Weg; M.C. van Engelen; G. Bor; L.A.P. Hoogenboom; M.W.F. Nielen (pp. 43-49).

This study deals with an automated data analysis strategy to pinpoint potential unknown compounds in full scan mass spectrometry (MS) experiments. Three examples of an untargeted metabolomics approach to contaminant analysis are given. By comparing a plant-oil based hormone cocktail to 90 plant oil samples ca. 25 compounds specific to the hormone cocktail could be detected. Five of these compounds were confirmed as steroid hormones. A comparison of a drink water sample from a farm to distillated water showed the presence of contaminants specific to this drink water sample. A grass sample, which was known to give a false positive result in a DR-CALUX bioassay, was unexpectedly shown to contain an abnormal level of chrysene, which was obviously not eliminated during clean-up.

Keywords: Metabolomics; Metalign; Contaminants; Hormone; Contaminants; Mass spectrometry

Use of statistical methods to find the polysaccharide structural characteristics and the relationships between monosaccharide composition ratio and macrophage stimulatory activity of regionally different strains of Lentinula edodes by Tiffany Chien Ting Lo; Yi Huei Jiang; Anne Lien Ju Chao; Cheng Allen Chang (pp. 50-56).

Multiple linear regression analysis was used to deduce the correlation between the monosaccharide composition ratios of 10 regionally different strains of Lentinula edodes and their in vitro macrophage stimulatory activities. Arabinose, xylose, mannose and galactose were identified as the monosaccharides that could be related to macrophage stimulatory activities. Additional principal component analysis and factor analysis methods were used to treat the same monosaccharide composition ratio data and the compositions of arabinose, xylose, mannose and galactose were found to be important. Interestingly, glucose, although presented in large compositions in all strains presumably forms the backbone of the polysaccharide structures, is not selected as the determinant factor for either structural characteristics or that of the in vitro macrophage stimulatory activities.

Keywords: Lentinula edodes; Polysaccharide; Multiple linear regression analysis; Principal component analysis; Factor analysis; Macrophage stimulatory activity

Assessment of quality performance parameters for straight line calibration curves related to the spread of the abscissa values around their mean by Jacques O. De Beer; Thomas R. De Beer; Leo Goeyens (pp. 57-65).

In validation of quantitative analysis methods, knowledge of the response function is essential as it describes, within the range of application, the existing relationship between the response (the measurement signal) and the concentration or quantity of the analyte in the sample. The most common response function used is obtained by simple linear regression, estimating the regression parameters slope and intercept by the least squares method as general fitting method. The assumption in this fitting is that the response variance is a constant, whatever the concentrations within the range examined.The straight calibration line may perform unacceptably due to the presence of outliers or unexpected curvature of the line. Checking the suitability of calibration lines might be performed by calculation of a well-defined quality coefficient based on a constant standard deviation.The concentration value for a test sample calculated by interpolation from the least squares line is of little value unless it is accompanied by an estimate of its random variation expressed by a confidence interval. This confidence interval results from the uncertainty in the measurement signal, combined with the confidence interval for the regression line at that measurement signal and is characterized by a standard deviation s x₀ calculated by an approximate equation. This approximate equation is only valid when the mathematical function, calculating a characteristic value g from specific regression line parameters as the slope, the standard error of the estimate and the spread of the abscissa values around their mean, is below a critical value as described in literature.It is mathematically demonstrated that with respect to this critical limit value for g, the proposed value for the quality coefficient applied as a suitability check for the linear regression line as calibration function, depends only on the number of calibration points and the spread of the abscissa values around their mean.

Keywords: Linear regression; Straight line calibration; Quality coefficient; g; -value; Spread of; x; -values around their mean; QC-; g; relationship

Rapid detection of Escherichia coli O157:H7 spiked into food matrices by Lisa C. Shriver-Lake; Stephanie Turner; Chris R. Taitt (pp. 66-71).

Food poisoning causes untold discomfort to many people each year. One of the primary culprits in food poisoning is Escherichia coli O157:H7. While most cases cause intestinal discomfort, up to 7% of the incidences lead to a severe complication called hemolytic uremic syndrome which may be fatal. The traditional method for detection of E. coli O157:H7 in cases of food poisoning is to culture the food matrices and/or human stool. Additional performance-based antibody methods are also being used. The NRL array biosensor was developed to detect multiple antigens in multiple samples with little sample pretreatment in under 30min. An assay for the specific detection of E. coli O157:H7 was developed, optimized and tested with a variety of spiked food matrices in this study. With no sample pre-enrichment, 5×10³cellsmL⁻¹ were detected in buffer in less than 30min. Slight losses of sensitivity (1–5×10⁻⁴cellmL⁻¹⁾ but not specificity occur in the presence of high levels of extraneous bacteria and in various food matrices (ground beef, turkey sausage, carcass wash, and apple juice). No significant difference was observed in the detection of E. coli O157:H7 in typical culture media (Luria Broth and Tryptic Soy Broth).

Keywords: Escherichia coli; O157:H7; Biosensor; Immunosensor; Food pathogen

Real-time monitoring of peptic and tryptic digestions of bovine β-casein using quartz crystal microbalance by Andreas Huenerbein; Christian E.H. Schmelzer; Reinhard H.H. Neubert (pp. 72-77).

In this study peptic and tryptic digestions of bovine β-casein were investigated using quartz crystal microbalance (QCM). β-Casein, which was used as a model protein, was immobilized on the surface of the QCM sensor where its degradation caused shifts in the resonant frequency. Atomic force microscopy was applied for the characterization of the protein layer. Different pH-values for peptic or tryptic digestions were chosen to visualize their effect on enzyme activity. Lower frequency shifts were observed at pH-values deviating from those at the maximum enzyme activity. In the case of the peptic digestion the frequency shift at pH 4 was more than 10 times smaller than those at pH 2. The frequency shifts for tryptic digestions at pH 5.4 and pH 6.4 were about two thirds compared to that obtained for the digestion at pH 7.4. The identification of peptides using MALDI-ToF mass spectrometry was used for verification of the proteolyses of the immobilized protein. Furthermore, it was shown that the QCM technique allows close observation of the effect of different pH-values on the immobilized casein layer. All in all, QCM facilitates the monitoring of the progress of enzymatic reactions in real-time.

Keywords: Quartz crystal microbalance; Milk protein; Proteolysis; Pepsin; Trypsin; Mass spectrometry

Detection and classification of gaseous sulfur compounds by solid electrolyte cyclic voltammetry of cermet sensor array by Kirsten E. Kramer; Susan L. Rose-Pehrsson; Mark H. Hammond; Duane Tillett; Holger H. Streckert (pp. 78-88).

Electrochemical sensors composed of a ceramic–metallic (cermet) solid electrolyte are used for the detection of gaseous sulfur compounds SO₂, H₂S, and CS₂ in a study involving 11 toxic industrial chemical (TIC) compounds. The study examines a sensor array containing four cermet sensors varying in electrode–electrolyte composition, designed to offer selectivity for multiple compounds. The sensors are driven by cyclic voltammetry to produce a current–voltage profile for each analyte. Raw voltammograms are processed by background subtraction of clean air, and the four sensor signals are concatenated to form one vector of points. The high-resolution signal is compressed by wavelet transformation and a probabilistic neural network is used for classification. In this study, training data from one sensor array was used to formulate models which were validated with data from a second sensor array. Of the 11 gases studied, 3 that contained sulfur produced the strongest responses and were successfully analyzed when the remaining compounds were treated as interferents. Analytes were measured from 10 to 200% of their threshold-limited value (TLV) according to the 8-h time weighted average (TWA) exposure limits defined by the National Institute of Occupational Safety and Health (NIOSH). True positive classification rates of 93.3, 96.7, and 76.7% for SO₂, H₂S, and CS₂, respectively, were achieved for prediction of one sensor unit when a second sensor was used for modeling. True positive rates of 83.3, 90.0, and 90.0% for SO₂, H₂S, and CS₂, respectively, were achieved for the second sensor unit when the first sensor unit was used for modeling. Most of the misclassifications were for low concentration levels (such 10–25% TLV) in which case the compound was classified as clean air. Between the two sensors, the false positive rates were 2.2% or lower for the three sulfur compounds, 0.9% or lower for the interferents (eight remaining analytes), and 5.8% or lower for clean air. The cermet sensor arrays used in this analysis are rugged, low cost, reusable, and show promise for multiple compound detection at parts-per-million (ppm) levels.

Keywords: Cermet; Sensor array; Pattern recognition; Gas sensor; Probabilistic neural network; Cyclic voltammetry

Zn(II) complex-based potentiometric sensors for selective determination of nitrate anion by Rakesh Kumar Mahajan; Ravneet Kaur; Hiroyuki Miyake; Hiroshi Tsukube (pp. 89-94).

Polymeric membranes containing new Zn(II) complexes as anion carriers were prepared for determination of nitrate anion present in water samples. Two Zn(II) complexes coordinated by neutral tetradentate ligands, N,N′-ethylene-bis( N-methyl-( S)-alanine methylamide) and N,N′-ethylene-bis( N-methyl-( S)-alanine dimethylamide), worked well as anion-selective carriers, while common phthalocyanine Zn(II) complex rarely responded to any anions. The combination of these new Zn(II) complexes with dioctylsebacate as a plasticizer particularly offered high sensing selectivity for nitrate anion. They exhibited near-Nernstian slopes in the wide linear concentration range of 5.0×10⁻⁵ to 1.0×10⁻¹M, and operated well in the wide pH range from 4 to 11 with the response time of less than 25s. The potentiometric selectivity coefficients were evaluated using the fixed interference method, indicating that the two Zn(II) complexes exhibited better selectivity for nitrate anion with respect to a wide variety of inorganic anions. Although chloride anion worked as an interfering species at a concentration higher than 1.0×10⁻³M, the new Zn(II) complex-based sensors were applicable in determination of the nitrate anion after adding silver sulfate to remove the chloride anion.

Keywords: Zn(II) complexes; Nitrate anion; Polymeric membranes; Anion-selective sensors

A highly selective and sensitive fluorescein-based chemodosimeter for Hg²⁺ ions in aqueous media by Xiao-Feng Yang; Yu Li; Quan Bai (pp. 95-100).

A new fluorescein-based chemodosimeter (II) for Hg²⁺ ion was designed and synthesized, and it displayed excellent selective and sensitive toward Hg²⁺ ion over other commonly metal ions in aqueous media.II was a colorless, non-fluorescent compound. Upon addition of Hg²⁺ to the solution ofII, the thiosemicarbazide moiety ofII would undergo an irreversible desulfurization reaction to form its corresponding oxadiazole (IV), a colorful and fluorescent product. During this process, the spirocyclic ring ofII was opened, causing instantaneous development of visible color and strong fluorescence emission in the range of 500–600nm. Based on the above mechanism, a fluorogenic Hg²⁺-selective chemodosimeter was developed. The fluorescence increase is linearly with Hg²⁺ concentration up to 1.0μmolL⁻¹ with a detection limit of 8.5×10⁻¹⁰molL⁻¹ (3 σ). Compared with the rhodamine-type chemodosimeter,II is more stable in aqueous media and exhibits higher sensitivity toward Hg²⁺. The findings suggest thatII will serve as a practical chemodosimeter for rapid detection of Hg²⁺ concentrations in realistic media.

Keywords: Hg; ²⁺; Fluorescein; Chemodosimeter; Fluorimetry

Independent component analysis of nanomechanical responses of cantilever arrays by Rick Archibald; Panos Datskos; Gerald Devault; Vincent Lamberti; Nickolay Lavrik; Don Noid; Michael Sepaniak; Pampa Dutta (pp. 101-105).

The ability to detect and identify chemical and biological elements in air or liquid environments is of far reaching importance. Performing this task using technology that minimally impacts the perceived environment is the ultimate goal. The development of functionalized cantilever arrays with nanomechanical sensing is an important step towards this goal. This report couples the feature extraction abilities of independent component analysis (ICA) and the classification techniques of neural networks to analyze the signals produced by microcantilever-array-based nanomechanical sensors. The unique capabilities of this analysis unleash the potential of this sensing technology to accurately identify chemical mixtures and concentrations. Furthermore, it is demonstrated that the knowledge of how the sensor array reacts to individual analytes in isolation is sufficient information to decode mixtures of analytes—a substantial benefit, significantly increasing the analytical utility of these sensing devices.

Keywords: Independent component analysis; Functionalized cantilever arrays; Neural networks; Nanomechanical sensors

Development of a gold nanoparticles based chemiluminescence imaging assay and its application by Lirong Luo; Zhujun Zhang; Lingyan Hou (pp. 106-111).

In this paper, a novel gold nanoparticles based protein immobilization method was designed. Biocomposites of gold nanoparticles and proteins were successfully coated on poly(methyl methacrylate) (PMMA) plates and polystyrene microtiter plates. The proteins could be immobilized on solid materials with high density and better bioactivity. Based on above design, chemiluminescence (CL) imaging assay for determination of H₂O₂ and recombinant human interleukin-6 (rHu IL-6) was developed. The linear range and the loading capability were greatly improved when compared with imaging assay performed with direct proteins immobilization. Under the selected experimental conditions, a linear relationship was obtained between the CL intensity and the concentration of H₂O₂ in the range of 1.0×10⁻⁶ to 1.0×10⁻⁴molL⁻¹, and rHu IL-6 in the range of 2.0–312.0pgmL⁻¹. The detection limits were 2×10⁻⁷molL⁻¹ (3 σ) for H₂O₂ and 0.5pgmL⁻¹ for rHu IL-6 with relative standard deviation of 3.8% for 3.0×10⁻⁵molL⁻¹ H₂O₂, and 4.4% for 39.0pgmL⁻¹ rHu IL-6. This method has been applied to the determination of rHu IL-6 in human serum with satisfactory results.

Keywords: Chemiluminescence; Imaging; Immobilization; Nanoparticles

Uniform molecularly imprinted microspheres and nanoparticles prepared by precipitation polymerization: The control of particle size suitable for different analytical applications by Keiichi Yoshimatsu; Kristina Reimhult; Anatol Krozer; Klaus Mosbach; Koji Sode; Lei Ye (pp. 112-121).

Molecularly imprinted polymers (MIPs) are being increasingly used as selective adsorbents in different analytical applications. To satisfy the different application purposes, MIPs with well controlled physical forms in different size ranges are highly desirable. For examples, MIP nanoparticles are very suitable to be used to develop binding assays and for microfluidic separations, whereas MIP beads with diameter of 1.5–3μm can be more appropriate to use in new analytical liquid chromatography systems. Previous studies have demonstrated that imprinted microspheres and nanoparticles can be synthesized using a simple precipitation polymerization method. Despite that the synthetic method is straightforward, the final particle size obtained has been difficult to adjust for a given template. In this work, we initiated to study new synthetic conditions to obtain MIP beads with controllable size in the nano- to micro-meter range, using racemic propranolol as a model template. Varying the composition of the cross-linking monomer allowed the particle size of the MIP beads to be altered in the range of 130nm to 2.4μm, whereas the favorable binding property of the imprinted beads remained intact. The chiral recognition sites were further characterized with equilibrium binding analysis using tritium-labeled ( S)-propranolol as a tracer. In general, the imprinted sites displayed a high chiral selectivity: the apparent affinity of the ( S)-imprinted sites for ( S)-propranolol was 20 times that of for ( R)-propranolol. Compared to previously reported irregular particles, the chiral selectivity of competitive radioligand binding assays developed from the present imprinted beads has been increased by six to seven folds in an optimized aqueous solvent.

Keywords: Molecular imprinting; Precipitation polymerization; Propranolol; Assay; Nanoparticles

Homogeneous, unmodified gold nanoparticle-based colorimetric assay of hydrogen peroxide by Zai-Sheng Wu; Song-Bai Zhang; Meng-Meng Guo; Chen-Rui Chen; Guo-Li Shen; Ru-Qin Yu (pp. 122-128).

An unmodified gold nanoparticle-based colorimetric assay system in homogeneous format has been developed using hydrogen peroxide (H₂O₂) as a model analyte. H₂O₂ is added to o-phenylenediamine/horseradish peroxidase solution, and allowed to react for 10min. Then, unmodified gold nanoparticles that serve as “reaction indicators” are added to the reaction solution. The resulting mixture color changes dramatically from red to blue. The reason is that azoaniline, a horseradish peroxidase-catalyzed oxidation product, induces the nanoparticle aggregation. Using this approach, H₂O₂ can be semiquantitatively determined over the concentration range of ∼4 orders of magnitude by the naked eye. If the observed peak intensity at 420nm is used for the construction of the calibration plot, hydrogen peroxide can be accurately determined down to concentration levels of 1.3×10⁻⁶M. Compared with the conventional electrochemical protocol, this sensing system offers several important advantages: (1) ability to be monitored by the naked eye, (2) avoiding the need of surface modification of electrodes or gold nanoparticles and (3) detection in homogeneous solution. It is worthy of note that this efficient and convenient strategy is also suitable for the detection of other species, such as glucose and cholesterol.

Keywords: Colorimetric assay; Gold nanoparticle aggregation; Horseradish peroxidase (HRP); o; -Phenylenediamine (OPD)

Characterization of electrokinetic gating valve in microfluidic channels by Guiseng Zhang; Wei Du; Bi-Feng Liu; Hideaki Hisamoto; Shigeru Terabe (pp. 129-135).

Electrokinetic gating, functioning as a micro-valve, has been widely employed in microfluidic chips for sample injection and flow switch. Investigating its valving performance is fundamentally vital for microfluidics and microfluidics-based chemical analysis. In this paper, electrokinetic gating valve in microchannels was evaluated using optical imaging technique. Microflow profiles at channels junction were examined, revealing that molecular diffusion played a significant role in the valving disable; which could cause analyte leakage in sample injection. Due to diffusion, the analyte crossed the interface of the analyte flow and gating flow, and then formed a cometic tail-like diffusion area at channels junction. From theoretical calculation and some experimental evidences, the size of the area was related to the diffusion coefficient and the velocity of analytes. Additionally, molecular diffusion was also believed to be another reason of sampling bias in gated injection.

Keywords: Electrokinetic gating; Microfluidic chip; Valving; Leakage

Determination of alternative and conventional chelating agents as copper(II) complexes by capillary zone electrophoresis—The first use of didecyldimethylammonium bromide as a flow reversal reagent by Pirkko-Leena Laamanen; Rose Matilainen (pp. 136-144).

A capillary zone electrophoresis (CZE) method for analyzing 11 chelating agents [β-alaninediacetic acid (β-ADA), trans-1,2-diaminocyclohexane- N, N, N′, N′-tetraacetic acid (CDTA), diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), N-(2-hydroxyethyl)ethylenediamine- N, N′, N′-triacetic acid (HEDTA), N-(2-hydroxyethyl)iminodiacetic acid (HEIDA), iminodiacetic acid (IDA), methylglycinediacetic acid (MGDA), nitrilotriacetic acid (NTA), 1,3-diaminopropane- N, N, N′, N′-tetraacetic acid (PDTA) and triethylenetetraaminehexaacetic acid (TTHA)] as negatively charged copper(II) complexes has been established. Both conventional and alternative chelating agents were included in this study, because they are used side by side in industrial applications. In this study, didecyldimethylammonium bromide (DMDDAB) was successfully used as a flow reversal reagent for the first time in an aqueous CZE method based on phosphate BGE with UV spectrophotometric detection. In addition this new flow modifier was compared to common TTAB. Method development was done using a fused silica capillary (61cm×50μm i.d.). The optimized BGE was a 105mmolL⁻¹ phosphate buffer with TTAB or DMDDAB in the concentration 0.5mmolL⁻¹ at pH 7.1. The measurements were done with −20kV voltage using direct UV detection at 254nm. In both CZE methods all 11 analyte zones were properly separated (resolutions ≥2.4), and the calibrations gave excellent correlation coefficients (≥0.998; linear range tested 0.5–2.0mmolL⁻¹). The limits of detection were ≤34 and ≤49μmolL⁻¹ with the method of DMDDAB and TTAB, respectively. A clear benefit of both methods was the short analysis time; all 11 complexes were detected in less than 6 and 5.5min with the methods of TTAB and DMDDAB, respectively. The two methods were tested with dishwashing detergents and paper mill wastewater samples and proved to be suitable for practical use.

Keywords: Chelating agents; Capillary zone electrophoresis; Didecyldimethylammonium bromide; Aminopolycarboxylic acids; Hydroxycarboxylic acids

Simple and rapid determination of biogenic amines in wine by liquid chromatography–electrospray ionization ion trap mass spectrometry by Silvia Millán; M. Carmen Sampedro; Nora Unceta; M. Aranzazu Goicolea; Ramón J. Barrio (pp. 145-152).

A rapid liquid chromatographic–electrospray ionisation ion trap mass spectrometry (LC-ESI-ITMS) method has been developed for the routine analysis of eight of the most oenologically important biogenic amines in wine without any sample pre-treatment. The method involves addition of heptylamine as an internal standard (IS) and the direct injection of filtered wine samples previously diluted with ultra high purity (UHP) water. The full-scan MS–MS spectra and the identical retention times to those of reference standards were used for unequivocal identification of the analytes. For most amines, the most abundant ions were derived from the loss of an ammonia group, while in the case of spermine and the I.S. the major product ions arose from the loss of 1,3-propyldiamine and the production of adduct with water, respectively. Detection was achieved in positive ionisation with an ion trap mass spectrometer operating in multiple-reaction monitoring (MRM) mode. The method allowed accurate determination of the analytes in the range 0.5–40ngmL⁻¹. Within-day and between-day relative standard deviation percentages were <8% and <12%, respectively. The overall process was successfully applied to identify and quantify biogenic amines in Rioja red wines. The new method is sensitive, rapid, cheap and less labour intensive.

Keywords: Biogenic amines; Wine; LC–ESI-MS/MS; Ion trap analyser

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations by Demetra Kyriakides; Irene Panderi (pp. 153-159).

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C₁₈ analytical column (250mm×4.6mm i.d., 5μm particle size); the mobile phase consisted of 0.005M tetrabutylammonium hydroxide and 0.005M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00mLmin⁻¹. The ultraviolet (UV) detector was operated at 262nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95min, respectively. The calibration graph was ranged from 2.50 to 20.00μgmL⁻¹, while detection and quantitation limits were found to be 0.48 and 1.61μgmL⁻¹, respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, % E_r, was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.

Keywords: Risedronate; Magnesium ascorbyl phosphate; Ion-pair liquid chromatography; Pharmaceutical dosage forms; Stability indicating

Rapid chiral separation and impurity determination of levofloxacin by ligand-exchange chromatography by Hongyuan Yan; Kyung Ho Row (pp. 160-165).

A sensitive, simple, and accurate method for determination of levofloxacin and its ( R)-enantiomer was developed to determine the chiral impurity of levofloxacin in Cravit Tablets material by ligand-exchange high performance liquid chromatography. The effects of different kinds of ligands, concentration of ligands in mobile phase, organic modifier, pH of mobile phase, and temperature on enantioseparation were investigated and evaluated. Chiral separation was performed on a conventional C₁₈ column, where the mobile phase consisted of a methanol–water solution (containing10mmolL⁻¹l-leucine and 5mmolL⁻¹ copper sulfate) (88:12, v/v) and its flow-rate was set at 1.0mLmin⁻¹. The conventional C₁₈ column offers baseline separation of two enantiomers with a resolution of 2.4 in less than 20min. Thermodynamic data (ΔΔ H and ΔΔ S) obtained by Van’t Hoff plots revealed the chiral separation is an enthalpy-controlled process. The standard curves showed excellent linearity over the concentration range from 0.5 to 400mgL⁻¹ for levofloxacin and its ( R)-enantiomer. The linear correlation equations are: y=1.33×10⁵ x+6297 ( r=0.9991) and y=1.34×10⁵ x+3565 ( r=0.9997), respectively. The relative standard deviation (RSD) of the method was below 2.3% ( n=3).

Keywords: Enantioseparation; Levofloxacin; Impurity; Ligand-exchange chromatography

Determination of trace trichlorfon by high performance liquid chromatography with UV detection based on its catalytic effect on sodium perborate oxidizing benzidine by Hai-Zhen Zhu; Yan-Mei Cui; Xiu-Wen Zheng; Hang-Ru Han; Ming-Min Yang (pp. 166-171).

Trichlorfon has the capacity to catalyze the oxidation of benzidine (4,4′-diamino-biphenyl) to 4-amino-4′-nitro biphenyl in the presence of sodium perborate. The product of the catalyzed reaction was validated by LC–MS method. Reversed-phase high performance liquid chromatography with 365nm UV detection was used for separation and quantification of 4-amino-4′-nitro biphenyl. It can be proven there is a linear relationship between the peak areas of 4-amino-4′-nitro biphenyl and trichlorfon in the concentration range of 0.02–0.5mgL⁻¹ ( r=0.9988). Limit of detection was 2.0μgL⁻¹. A method for the indirect determination of trichlorfon using HPLC was developed based on catalytic effect of trichlorfon. Method validation was performed on samples spiked at three levels (0.5, 1.0, 1.5mgkg⁻¹), the recoveries ranged from 67.5 to 82.1%, with relative standard deviations between 4.5 and 7.3%.0.01molL⁻¹ sodium dodecyl sulphate (SDS) solution was used to extract trichlorfon from samples and solid-phase extraction was used to isolate and concentrate trichlorfon in SDS solution. The recoveries of trichlorfon obtained with percolating the extraction through a SPE system were essentially in agreement with those obtained by liquid–liquid extraction. This new isolation technique decreases the use of toxic solvents and satisfies the requirements of Green Analytical Chemistry.

Keywords: Catalytic effect; Micellar extraction; Trichlorfon; Benzidine; Sodium perborate

Gas chromatography/mass spectrometry characterization of historical varnishes of ancient Italian lutes and violin by J.P. Echard; C. Benoit; J. Peris-Vicente; V. Malecki; J.V. Gimeno-Adelantado; S. Vaiedelich (pp. 172-180).

The organic constituents of historical vanishes from two ancient Italian lutes and a Stradivari violin, kept in the Musée de la musique in Paris, have been characterized using gas chromatography–mass spectrometry. Results have been compared with the chromatograms and mass spectra of recent as well as old naturally aged reference materials. The three historical varnishes analyzed have been shown to be oil varnishes, probably mixtures of linseed oil with resins. Identification of diterpenoids and triterpenoids compounds, and of the resins that may have been ingredients of the varnishes, are discussed in this paper.

Keywords: Gas chromatography/mass spectrometry; Musical instruments; Varnish; Stradivari; Natural resins; Colophony; Venice turpentine; Elemi

Use of coacervates for the on-site extraction/preservation of polycyclic aromatic hydrocarbons and benzalkonium surfactants by Noelia Luque; Soledad Rubio; Dolores Pérez-Bendito (pp. 181-188).

The suitability of coacervates for the preservation of organic pollutants after their extraction from water samples was investigated for the first time. Acid-induced sodium dodecanesulfonic acid (SDSA) micelle-based coacervates were selected for this purpose. Their capacity to preserve benzalkonium homologue (C₁₂, C₁₄ and C₁₆) surfactants (BASs) and different polycyclic aromatic hydrocarbons (PAHs) [benzo( a)pyrene (BaP), benzo( b)fluoranthene (BbF), benzo( k)fluoranthene (BkF), benzo( ghi)perylene (B ghiP), benzo( a)anthracene (BaA) and indene(1,2,3- c- d)pyrene (IP)] was investigated. BASs and PAHs were efficiently extracted by the coacervate by formation of mixed aggregates and hydrophobic interactions, respectively. Their stability into the coacervate was investigated under three temperature conditions (room temperature, 4°C and −20°C) and two hydrochloric acid concentrations (3.75M and 4.2M), which was used to induce coacervation. No losses were observed during at least 3 months at the different experimental conditions tested. The increase of the temperature up to 35°C for a month did not affect the stability of the target compounds. No influence of the water matrix (distilled, river or wastewater) on the stabilization of BASs and PAHs was observed. The high-stabilizing capacity of the coacervate for the target compounds and its low volume make easy the transport and storage of analytes.

Keywords: Polycyclic aromatic hydrocarbons; Benzalkonium surfactants; Preservation; Micelle-based coacervates; Monitoring programs

An ionic liquid as a solvent for headspace single drop microextraction of chlorobenzenes from water samples by Lorena Vidal; Elefteria Psillakis; Claudia E. Domini; Nuria Grané; Frank Marken; Antonio Canals (pp. 189-195).

A headspace single-drop microextraction (HS-SDME) procedure using room temperature ionic liquid and coupled to high-performance liquid chromatography capable of quantifying trace amounts of chlorobenzenes in environmental water samples is proposed. A Plackett–Burman design for screening was carried out in order to determine the significant experimental conditions affecting the HS-SDME process (namely drop volume, aqueous sample volume, stirring speed, ionic strength, extraction time and temperature), and then a central composite design was used to optimize the significant conditions. The optimum experimental conditions found from this statistical evaluation were: a 5μL microdrop of 1-butyl-3-methylimidazolium hexafluorophosphate, exposed for 37min to the headspace of a 10mL aqueous sample placed in a 15mL vial, stirred at 1580rpm at room temperature and containing 30% (w/v) NaCl. The calculated calibration curves gave a high level of linearity for all target analytes with correlation coefficients ranging between 0.9981 and 0.9997. The repeatability of the proposed method, expressed as relative standard deviation, varied between 1.6 and 5.1% ( n=5). The limits of detection ranged between 0.102 and 0.203μgL⁻¹. Matrix effects upon extraction were evaluated by analysing spiked tap and river water as well as effluent water samples originating from a municipal wastewater treatment plant.

Keywords: Chlorinated benzenes; Headspace single-drop microextraction (HS-SDME); Solvent microextraction; Ionic liquid; Water analysis; High-performance liquid chromatography (HPLC)

Solid phase selective separation and preconcentration of Cu(II) by Cu(II)-imprinted polymethacrylic microbeads by Ivanka Dakova; Irina Karadjova; Ivo Ivanov; Ventsislava Georgieva; Bisera Evtimova; George Georgiev (pp. 196-203).

Ion-imprinted polymer (IIP) particles are prepared by copolymerization of methacrylic acid as monomer, trimethylolpropane trimethacrylate as crosslinking agent and 2,2′-azo-bis-isobutyronitrile as initiator in the presence of Cu(II), a Cu(II)–4-(2-pyridylazo)resorcinol (Cu(II)–PAR) complex, and PAR only. A batch procedure is used for the determination of the characteristics of the Cu(II) solid phase extraction from the IIP produced. The results obtained show that the Cu(II)–PAR IIP has the greatest adsorption capacity (37.4μmolg⁻¹ of dry copolymer) among the IIPs investigated. The optimal pH value for the quantitative preconcentration is 7, and full desorption is achieved by 1M HNO₃. The selectivity coefficients ( S_Cu/Me) for Me=Ni(II), Co(II) are 45.0 and 38.5, respectively. It is established that Cu(II)–PAR IIPs can be used repeatedly without a considerable adsorption capacity loss. The determination of Cu(II) ions in seawater shows that the interfering matrix does not influence the preconcentration and selectivity values of the Cu(II)–PAR IIPs. The detection and quantification limits are 0.001μmolL⁻¹ (3 σ) and 0.003μmolL⁻¹ (6 σ), respectively.

Keywords: Solid phase extraction; Ion-imprinted polymer; Preconcentration; Selectivity coefficients; Flame atomic absorption spectrometry; Seawater

Determination of trace amounts of lead, arsenic, nickel and cobalt in high-purity iron oxide pigment by inductively coupled plasma atomic emission spectrometry after iron matrix removal with extractant-contained resin by Yuyu Xu; Jianfeng Zhou; Guoxin Wang; Jinfan Zhou; Guanhong Tao (pp. 204-209).

Inductively coupled plasma atomic emission spectrometry (ICP-AES) was applied to the determination of lead, arsenic, nickel and cobalt in high-purity iron oxide pigment. Samples were dissolved with hydrochloric acid and hydrogen peroxide. The digest was passed through a column, which was packed with a polymer resin containing a neutral organophosphorus extractant, tri- n-butylphosphate. Iron was sorbed selectively on the resin and the analytes of interest passed through the column, allowing the effective separation of them from the iron matrix. Conditions of separation were optimized. The detection limits (3 σ) in solution were 10, 40, 7 and 5μgL⁻¹, and in pigment were 0.2, 0.8, 0.14 and 0.1mgkg⁻¹ for lead, arsenic, cobalt and nickel, respectively. The recoveries ranged from 95% to 107% when sample digests were spiked with 5μg of the analytes of interest, and relative standard deviations ( n=6) were 1.5–17.6% for the determination of the spiked samples. The method was successfully applied to the determination of trace amounts of these elements in high-purity iron oxide pigment samples.

Keywords: High-purity iron oxide; Trace elements; Inductively coupled plasma atomic emission spectrometry; Matrix removal; Extractant-contained resin

Determination of traces of rubidium in high purity cesium chloride by electrothermal atomic absorption spectrometry (ETAAS) using boric acid as a modifier by K. Dash; S. Thangavel; S.C. Chaurasia; J. Arunachalam (pp. 210-214).

The use of boric acid as a modifier for the determination of trace amount of rubidium in high purity cesium chloride matrix by electrothermal atomic absorption is described. It was found that the negative influence of the chloride matrix could not be eliminated using stabilized temperature platform (STPF) alone. Due to the high dissociation energy ( D₀=427kJmol⁻¹) of rubidium chloride, it was difficult to dissociate in the gas phase and hence is lost. Elimination of interferences was achieved by the addition of boric acid as a chemical modifier. Diluted cesium chloride samples (5%, m/v) were analyzed applying the standard addition method. The characteristic mass of 24pg was obtained. The detection limit of the proposed method is around 26ngg⁻¹. The developed method was applied to the determination of traces of rubidium in high purity cesium chloride samples. The data obtained by this method were in good agreement with those obtained by other independent method like FAAS.

Keywords: Chloride interference; Rubidium; Cesium chloride; Boric acid modifier

Near-infrared diffuse reflectance spectroscopy and neural networks for measuring nutritional parameters in chocolate samples by J. Moros; F.A. Iñón; S. Garrigues; M. de la Guardia (pp. 215-222).

A rapid and non-destructive method has been developed for the characterization of chocolate samples based on diffuse reflectance near-infrared Fourier transform spectroscopy (DRIFTS) and artificial neural networks (ANNs). This methodology provides a potentially useful alternative to time-consuming chemical methods of analysis. To assess its utility, 36 chocolate samples purchased from the Spanish market were analyzed for the determination of the main nutritional parameters like carbohydrates, fat, proteins, energetic value and cocoa content.Direct triplicate measurements of each sample were carried out by DRIFTS. Cluster hierarchical analysis was used for selecting calibration and validation data sets, resulting in a calibration set comprised of 19 samples and a validation data set of 17 samples. As it is common the presence of non-linear effect in reflectance spectroscopy, ANNs was chosen for data pretreatment. The root-mean-square error of prediction (RMSEP) values obtained for carbohydrates, fat, energetic value and cocoa were 1.0% (w/w), 1.0% (w/w), 50kJ(100g)⁻¹ and 1.4%, respectively. The mean difference ( d x–_y) and standard deviation of mean differences ( s x–_y) of the carbohydrates, fat, proteins content, energetic value and cocoa content were 0.9 and 2.4% (w/w), 0.2 and 1.0% (w/w), 9.1 and 50kJ(100g)⁻¹, and −0.5 and 1.4%, respectively. The maximum relative error for the prediction (QC) of any of these parameters for a new sample did not exceed 5.2%.

Keywords: Carbohydrates; Fat; Protein; Energetic value; Chocolates; Near-infrared reflectance spectroscopy; Neural networks

Automatic alignment of infrared video frames for equipment leak detection by Liming Zhou; Yousheng Zeng (pp. 223-227).

Petroleum refining and petrochemical industries start using infrared (IR) cameras to detect volatile organic compounds (VOC) leaking out of process equipment. However, further quantitative processing of these video image data or automatic recognition of VOC plumes are hindered by unaligned video frames owing to the slight vibrations of the camera. An automatic method is developed to align the IR video frames as a preprocessing procedure for other possible video processing methods. The alignment method is based on a two-dimensional spatial Fourier transform. The accuracy can reach fractional pixels in estimation of translational shift and 1–2° for rotational shift. Temporal Fourier transform of actual industrial tests of IR videos is performed with both unaligned and aligned video frames. The results indicate that only after the alignment of the video frames, the camera motion interferences on VOC plume identification can be eliminated or minimized, and the VOC plume can be identified through investigating the characteristic flickering frequency power in the temporal Fourier transform. This alignment method provides a useful tool for IR or other optical video image data preprocessing purposes.

Keywords: Video frame alignment; Volatile organic compounds leak detection; Image registration; Leak detection and repair

Interference modelling, experimental design and pre-concentration steps in validation of the Fenton's reagent for pesticides determination by Miren Ostra; Carlos Ubide; Juan Zuriarrain (pp. 228-235).

The determination of atrazine in real samples (commercial pesticide preparations and water matrices) shows how the Fenton's reagent can be used with analytical purposes when kinetic methodology and multivariate calibration methods are applied. Also, binary mixtures of atrazine–alachlor and atrazine–bentazone in pesticide preparations have been resolved. The work shows the way in which interferences and the matrix effect can be modelled. Experimental design has been used to optimize experimental conditions, including the effect of solvent (methanol) used for extraction of atrazine from the sample. The determination of pesticides in commercial preparations was accomplished without any pre-treatment of sample apart from evaporation of solvent; the calibration model was developed for concentration ranges between 0.46 and 11.6×10⁻⁵molL⁻¹ with mean relative errors under 4%. Solid-phase extraction is used for pre-concentration of atrazine in water samples through C₁₈ disks, and the concentration range for determination was established between 4 and 115μgL⁻¹approximately. Satisfactory results for recuperation of atrazine were always obtained.

Keywords: Fenton's reagent; Atrazine; Pesticide binary mixtures; Interference modelling; Experimental design; Solid-phase extraction

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Analytica Chimica Acta (v.584, #1)

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Analytica Chimica Acta (v.584, #1)