Analytica Chimica Acta (v.533, #2)

Contents (iii-iv).

The development of electrochemiluminescence (ECL) detection for capillary electrophoresis (CE) is critically reviewed. After a simple discussion of the mechanism of ECL concerning in CE–ECL system and CE mode, special attention is focused on the instrumental design, mainly including addition of reagent, field decoupler, and ECL efficiency. Application of the CE–ECL technique was also given.
Keywords: Capillary electrophoresis; Electrochemiluminescence; Review;

Stability-indicating assay using capillary zone electrophoresis for an azaphenothiazine in an ointment formulation by Bassem Baalbaki; Edmond Cheble; Gustave Nguema; Huguette Fabre (121-125).
A stability-indicating assay using a capillary zone electrophoresis method is reported for the determination of isothipendyl in an ointment formulation. Sample preparation consisted of a simple dissolution of the ointment in an internal standard (3-aminobenzoic acid hydrochloride) solution. Separation was carried out in a short fused silica capillary using a pH 2.5 phosphate buffer as electrolyte. After introduction of the sample solution into the capillary, the compounds were separated as cations and detected within 3 min. The procedure was validated following the guidelines of the International Conference on Harmonisation. The method was found to be specific in relation to potential degradation products and excipients. Linearity determined between 50 and 150% of the target concentration was satisfactory (r 2  = 0.999). Recovery studies from an analytical placebo spiked with the analyte at five concentration levels (50–150% of the target concentration) on each of 3 days ranged from 103.5 to 99.2%. The repeatability of the entire procedure (n  = 7) was better than 1.0%. The detection limit was estimated to be approximately 0.5 mg l−1.
Keywords: Azaphenothiazine; Capillary zone electrophoresis; Isothipendyl; Phenothiazine; Ointment formulation; Validation;

A new capillary zone electrophoresis (CZE) method for the separation of omeprazole enantiomers has been developed. Methyl-β-cyclodextrin (methyl-β-CD) was chosen as the chiral selector, and several parameters, such as cyclodextrin structure and concentration, buffer concentration, pH, and capillary temperature were investigated in order to optimize separation and run times. Analysis times, shorter than 8 min were found using a background electrolyte solution consisting of 40 mM phosphate buffer adjusted to pH 2.2, 30 mM β-cyclodextrin and 5 mM sodium disulphide, hydrodynamic injection, and 15 kV separation voltage. Detection limits were evaluated on the basis of baseline noise and were established 0.31 mg/l for the omeprazole enantiomers. The proposed method was applied to five pharmaceutical preparations with recoveries between 84 and 104% of the labeled contents.
Keywords: Omeprazole; Chiral separation; Cyclodextrin; Capillary zone electrophoresis;

Detection of DNA damaging agents using layer-by-layer assembly by Gilbert E. Pacey; Sean D. Puckett; Long Cheng; Sheerin Khatib-Shahidi; James A. Cox (135-139).
The layer-by-layer (LBL) electrostatic deposition method was used to fabricate multilayer films of pentaerythritol-based metallodendrimer with RuII terpyridine subunits (RuDen) that has a positive charge and ds-DNA (ds, double-stranded) that has a negative charge due to its phosphate backbone. Evidence of assembly was obtained by fabrication of (DNA | RuDen) n on quartz treated with poly(diallyldimethylammonium chloride), PDDA. The absorbance at 263 nm varied linearly with n in the range 1–6. For electrochemical monitoring of damage by styrene oxide, the assembly was on a glassy carbon electrode that was coated with a monolayer of aminobenzoic acid. The measurement was based upon the RuDen-catalyzed oxidation of sites, e.g. guanine, that are exposed when ds-DNA is damaged. The peak current at 1.07 V versus Ag | AgCl in square wave voltammetry increases with incubation time for 30 min. The process was also monitored by the shift in the spectrum of a long period grating (LPG) fiber coated with (DNA | RuDen)5. A typical shift, which is due to changes in the refractive index of the coating, was 0.3 and 1.8 nm for 5 and 30 min exposures, respectively, using an algorithm that can measure a shift of 10−4  nm.
Keywords: Modified electrodes; Electrocatalysis; Long period grating; Refractive index; Nanostructured coating;

On-site monitoring of trace levels of free manganese in sea water via sonoelectroanalysis using a boron-doped diamond electrode by Alexander Goodwin; Andrew L. Lawrence; Craig E. Banks; Frédéric Wantz; Dario Omanović; Šebojka Komorsky-Lovrić; Richard G. Compton (141-145).
Cathodic stripping voltammetry at a boron-doped diamond electrode utilising an insonated accumulation protocol is shown to be possible for the on-site trace detection of free manganese in seawater samples. No sample pre-treatment is required with the application of ultrasound providing a sensitive and selective protocol for the analysis of manganese. The protocol is used in the evaluation of manganese in seawater samples taken near the site of a former ferromanganese factory in the town Šibenik, Croatia.
Keywords: Manganese; Cathodic stripping voltammetry; Boron-doped diamond electrodes; Sea water; Environmental monitoring;

Ensemble methods and data augmentation by noise addition applied to the analysis of spectroscopic data by M.J. Sáiz-Abajo; B.-H. Mevik; V.H. Segtnan; T. Næs (147-159).
Near-infrared spectroscopy has gained great acceptance in the industry due to its multiple applications and versatility. Sometimes, however, the construction of accurate and robust calibration models involves the collection of a large number of samples with related reference analysis that can complicate and prolong the calibration stage.In this paper, ensemble methods and data augmentation by noise simulation have been applied to spectroscopic data in combination with PLSR to obtain robust models able to handle different types of perturbations likely to affect NIR data. Several types of noise have been investigated as well as different ensemble methods focused on obtaining robust PLS models able to predict both the original and the perturbed test data.The suitability of ensemble methods to perform robust calibration models has been investigated and compared to extended multiplicative signal correction (EMSC) and other calibration approaches in a real case of temperature compensation. Extended multiplicative signal correction (EMSC) and ensemble methods seem to be the most appropriate methods yielding the best results in terms of accuracy and prediction ability with a reduced calibration data set.
Keywords: Ensemble methods; Data augmentation; Noise simulation; PLSR; EMSC; NIRS;

Evaluation of chemometric techniques and artificial neural networks for cancer screening using Cu, Fe, Se and Zn concentrations in blood serum by Edwin A. Hernández-Caraballo; Francklin Rivas; Anna G. Pérez; Lué M. Marcó-Parra (161-168).
It is known that variations in the concentrations of certain trace elements in bodily fluids may be an indication of an alteration of the organism's normal functioning. Multivariate analysis of such data, and its comparison against proper reference values, can thus be employed as possible screening tests. Such analysis has usually been conducted by means of chemometric techniques and, to a lower extent, backpropagation artificial neural networks. Despite the excellent classification capacities of the latter, its development can be time-consuming and computer-intensive. Probabilistic artificial neural networks represent another attractive, yet scarcely evaluated classification technique which could be employed for the same purpose. The present work was aimed at comparing the performance of two chemometric techniques (principal component analysis and logistic regression) and two artificial neural networks (a backpropagation and a probabilistic neural network) as screening tools for cancer. The concentrations of copper, iron, selenium and zinc, which are known to play an important role in body chemistry, were used as indicators of physical status. Such elements were determined by total reflection X-ray fluorescence spectrometry in a sample of blood serum taken from individuals who had been given a positive (N  = 27) or a negative (N  = 32) diagnostic on various forms of cancer. The principal components analysis served two purposes: (i) initial screening of the data; and, (ii) reducing the dimension of the data space to be input to the networks. The logistic regression, as well as both artificial neural networks showed an outstanding performance in terms of distinguishing healthy (specificity: 90–100%) or unhealthy individuals (sensitivity: 100%). The probabilistic neural network offered additional advantages when compared to the more popular backpropagation counterpart. Effectively, the former is easier and faster to develop, for a smaller number of variables has to be optimized, and there are no risk of overtraining.
Keywords: Principal components analysis (PCA); Logistic regression; Artificial neural networks (ANNs); Backpropagation neural network (BpNN); Probabilistic neural network (PrNN); Cancer; Serum; Iron; Copper; Zinc; Selenium;

In this work, direct determination of lorazepam, an anxiolytic and sedative agent, in pharmaceutical formulations and biological fluids (urine and human plasma) was accomplished based on ultraviolet spectrophotometry (260–380 nm) using parallel factor analysis (PARAFAC) and partial least squares (PLS). The study was carried out in the pH range from 1.0 to 12.0 and with a concentration range from 0.50 to 8.75 μg ml−1 of lorazepam. Multivariate calibration models using PLS at different pH and PARAFAC were elaborated for ultraviolet spectra deconvolution and lorazepam quantitation. The best models for the system were obtained with PARAFAC and PLS at pH = 2.05 (PLS-PH2). The capabilities of the method for the analysis of real samples were evaluated by determination of lorazepam in pharmaceutical preparations and biological (urine and plasma) fluids with satisfactory results. The accuracy of the method, evaluated through the root mean square error of prediction (RMSEP), was 0.0429 for lorazepam with best calibration curve by PARAFAC and 0.0467 for lorazepam with PLS model at best pH. The protolytic equilibria of lorazepam at 25 °C and ionic strength of 0.1 M have also been determined spectrophotometrically. Protolytic equilibria of lorazepam were evaluated by DATAN program using the corresponding absorption spectra-pH data. The obtained pK a values of lorazepam are 1.54 and 11.61 for pK a1 and pK a2, respectively.
Keywords: Lorazepam; PARAFAC; PLS; DATAN; Pharmaceutical formulations; Biological fluids; Protolytic equilibria; Ultraviolet spectrophotometry;

This article describes a straightforward method for the quantification of thiol groups on the surface of chromatographic particles such as 3-mercaptopropyl silica gel. The method is based on a thiol-disulfide exchange reaction with 2,2′-dipyridyl disulfide (DPDS) reagent and spectrophotometric or HPLC-UV determination of the liberated 2-pyridyl thione. Linearity was obtained over the sulfhydryl concentration range of 0.02–0.5 μmol ml−1 for the spectrophotometric determination and 0.2–5 μmol ml−1 for the HPLC-UV measurement, with r 2 values of 0.9975 and 0.9981, respectively. The results were validated using elemental analysis as independent control method. The agreement between the results of both methods indicates that the sulfur of the thiol-modified silica particles is available as reactive sulfhydryls. The repeatability study indicated a higher precision for the DPDS method compared to the elemental analysis.
Keywords: Thiol; 2,2′-Dipyridyl disulfide; Thiol-disulfide exchange reaction; Chromatographic particles; Functionalized silica; Spectrophotometric; High performance liquid chromatography;

Standardization of antioxidant properties of honey by a combination of spectrophotometric/fluorimetric assays and chemometrics by Giangiacomo Beretta; Paola Granata; Maria Ferrero; Marica Orioli; Roberto Maffei Facino (185-191).
The aim of this work was to establish a solid platform of analytical information for the definition/standardization of the antioxidant properties of honey. We investigated first the antioxidant/radical scavenging capacity of 14 commercial honeys of different floral and geographic origin, using a battery of spectrophotometric tests: Folin-Ciocalteu assay for phenol content (PC), ferric reducing antioxidant power (FRAP assay) for total antioxidant activity, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay for antiradical activity, absorbance:450 (ABS450) for color intensity and one fluorimetric method: ORAC, oxygen reactive antioxidant capacity for the antilipoperoxidant activity. Then the data from different procedures were compared and analysed by multivariate techniques (correlation matrix calculation, principal component analysis (PCA)). Significant correlations were obtained for all the antioxidant markers (r ranging from 0.933 to 0.716), with antioxidant properties strictly correlated to the phenolic content and honey color intensity. PCA found different clusters of honey based on the antioxidant power and very likely also on chemical composition. The results of this study demonstrated that only through a combination of antioxidant testings, comparative analyses, and chemometric evaluation we can achieve a strictly rigorous guideline for the characterization of the antioxidant activity of honey, an invaluable tool for the understanding/demonstration of its antioxidants linked therapeutic efficacy.
Keywords: Standardization; Honey; Antioxidants; Optical methods; Multivariate analysis;

Metal speciation measurement by diffusive gradients in thin films technique with different binding phases by Weijia Li; Huijun Zhao; Peter R. Teasdale; Richard John; Feiyue Wang (193-202).
Since its invention in the mid-1990s, the diffusive gradients in thin films (DGT) technique has rapidly become one of the most promising in situ sampling techniques for trace metal measurement in natural waters. We investigated here the possibility of using DGT devices with different binding phases to determine different DGT labile fractions of Cd and Cu in laboratory solutions and in natural waters. Several binding phases were studied, including conventional Chelex 100 resin imbedded polyacrylamide hydrogel (Chelex) and several recently developed binding phases, poly(acrylamide-co-acrylic acid) (PAM-PAA) gel, poly(acrylamidoglycolic acid-co-acrylamide) (PAAG-PAM) gel, Whatman P81 cellulose phosphate ion-exchange membrane (P81), and poly(4-styrenesulfonate) (PSS) aqueous solution. Laboratory testing in metal solutions spiked with EDTA or humic acid suggested that all the DGT devices measured only free metal ions and inorganic metal complexes. Upon field testing at both freshwater and seawater sites it was found that the DGT labile metal concentrations measured by different binding phases can be significantly different, suggesting that the DGT labile metal fractions were dependent on binding strength of the binding phase. By designing binding phases that can compete with different natural water complexing ligands to varying extents, it is possible to use these different DGT devices to measure metal speciation in natural waters.
Keywords: In situ analysis; DGT; Metal speciation; Complexation; Waters;

In the present study, a microwave-assisted extraction method has been investigated for the extraction of persistent organochlorine pesticides from sediment in comparison to the classical Soxhlet extraction. The extraction procedure has been screened by a complete factorial design for statistically significant parameters. Thereafter, the identified parameters, composition of extraction solvent (mixtures of n-hexane–acetone), extraction temperature and time were optimized by a Box–Behnken design. Finally, the optimized microwave-assisted procedure has been validated by extraction of two different matrix reference materials, a sediment and a mallow powder. The data obtained for both materials were in good agreement with those obtained by Soxhlet extraction and published values. Special emphasis has been given to an accurate determination of p,p′-DDT, since DDT is known as instable during GC-injection. In order to evaluate the DDT-degradation, 13C12-p,p′-DDT-solutions were analyzed and the degradation rates during GC-injection were calculated. Furthermore, 13C12-p,p′-DDT was added to the sediment samples prior to extraction and the degradation rates during the optimization experiment have been investigated.
Keywords: Microwave-assisted extraction; Screening; Optimization; Validation; Sediment; Organochlorine pesticides; DDT degradation;

Through admittance measurements of two piezoelectric quartz crystals in parallel on one impedance analyzer and then non-linear fitting according to an equivalent circuit of two parallel Butterworth–Van Dyke circuits, we have simultaneously obtained accurate and precise impedance responses of two one-face sealed crystals to changes in solution density and viscosity, temperature, conductance, and/or electrode mass. A series of sucrose aqueous solutions, ferri-/ferrocyanide redox switching, hot water cooling, a series of NaClO4 aqueous solutions, bovine serum albumin adsorption and silver electrodeposition/stripping were selected as model systems for such purposes. Galvanostatic charging/discharging reactions at positive and negative poles in a Ni–Zn battery were synchronously monitored, with some quartz crystal microbalance (QCM) insights into the second reduction process of nickel hydroxide film. In all cases, the crystal immersion angle effect was found to be negligible. The present method as a versatile one is highly recommended for informative two-electrode monitoring of two concurrent chemical or biological events, or for check and/or compensation of effects due to solution density, viscosity, temperature and/or conductance during QCM researches.
Keywords: Simultaneous impedance analysis of two parallel piezoelectric quartz crystals; Density–viscosity effect; Temperature and conductance effects; Mass effects; Ni–Zn battery charging/discharging monitoring;

Author Index (225-226).