Analytica Chimica Acta (v.562, #1)

Contents (v-vi).

Detection of DNA hybridization was done on silica microbeads that were physically adsorbed onto glass surfaces. DNA oligonucleotide probes of approximately 20 mer size were immobilized on beads of 5 μm diameter after the microbeads were first silanized with 3-glycidoxypropyltrimethoxysilane. The suspension of silica microbeads in aqueous solution was spotted on the glass slides. After drying, the glass surface was washed with water and 1 × SSC buffer. Significant numbers of microbeads remained physically adsorbed onto the glass surfaces even after vigorous washing with buffer solution. After hybridization using approximately 200 mer PCR targets strands, the glass slides were scanned using a standard laser confocal fluorescence microscope microarray reader. The total time for completion of hybridization assays was less than 20 min for 100 nM samples of target oligonucleotide. The clinical utility of the method was demonstrated by detection of single base pair mutations in the survival motor neuron gene that is associated with the childhood disease Spinal Muscular Atrophy. The method proved to provide a readily adaptable strategy for immobilization of different probes in an array format, and provided for SNP detection on a disposable slide without cross contamination.
Keywords: Silica microbeads; DNA; Hybridization; SNP; Fluorescence; Array;

Solid-phase microextraction and gas chromatography with atomic emission detection for multiresidue determination of pesticides in honey by Natalia Campillo; Rosa Peñalver; Nerea Aguinaga; Manuel Hernández-Córdoba (9-15).
A method based on solid-phase microextraction (SPME) followed by gas chromatography with microwave-induced plasma atomic emission detection for determining 16 pesticides of different chemical families (organochlorines, organophosphorus compounds and pyrethrins) in honey is proposed. Parameters affecting the sample enrichment step, such as sample mass, ionic strength, absorption and desorption times and temperatures, were carefully optimized in the direct immersion mode. Element-specific detection and quantification was carried out by monitoring the chlorine (479 nm), bromine (478 nm) and sulphur (181 nm) emission lines, which provided nearly specific chromatograms. The matrix effect was evaluated for samples of different floral origin, it being concluded that standard addition calibration was required for quantification purposes. The detection limits ranged from 0.02 to 10 ng g−1, depending on the compound and the honey sample under analysis. The method is reliable and can be considered useful for routine monitoring. None of the honey samples analyzed contained the studied compounds at concentrations above the corresponding detection limits.
Keywords: Food analysis; Honey; Pesticides; Solid-phase microextraction (SPME); Gas chromatography–atomic emission detection (GC–AED);

We developed a sensitive and useful method for the determination of five fluoroquinolones (FQs), enoxacin, ofloxacin, ciprofloxacin, norfloxacin, and lomefloxacin in environmental waters, using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–tandem mass spectrometry (LC/MS/MS). These compounds were analysed within 7 min by high-performance liquid chromatography (HPLC) using a CAPCELL PAK C8 column and aqueous ammonium formate (pH 3.0, 5 mM)/acetonitrile (85/15, v/v) at a flow rate of 0.2 mL/min. Electrospray ionization conditions in the positive ion mode were optimized for MS/MS detection. In order to optimize the extraction of FQs, several in-tube SPME parameters were examined. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 μL of sample at a flow-rate of 150 μL/min, using a Carboxen 1010 PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase. Using the in-tube SPME LC/MS/MS method, good linearity of the calibration curve (r  ≥ 0.997) was obtained in the concentration range from 0.1 to 10 ng/mL for all compounds examined. The limits of detection (S/N = 3) of the five FQs ranged from 7 to 29 pg/mL. The in-tube SPME method showed 60–94-fold higher sensitivity than the direct injection method (5 μL injection). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several surface waters and wastewaters were collected from the area around Asahi River, and ofloxacin was detected in wastewater samples of a sewage treatment plant and other two hospitals at 17.5–186.2 pg/mL. The recoveries of FQs spiked into river water were above 81% for a 0.1 or 0.2 ng/mL spiking concentration, and the relative standard deviations were below 1.9–8.6%.
Keywords: In-tube solid-phase microextraction; Automated sample preparation; Liquid chromatography–tandem mass spectrometry; Fluoroquinolone antibacterial agents; Environmental water samples;

A new analytical procedure was developed using headspace solid-phase microextraction (HS-SPME) for the simultaneous determination of sorbic and benzoic acids in beverages. The sample were processed depending on their nature, either only diluted with water, or treated with a NaOH solution and filtered through a 0.45-μm membrane filter. The samples were heated in a vial in the presence of sulfuric acid and anhydrous sodium sulfate and the analytes were collected from the headspace by using a 65-μm polydimethylsiloxane–divinylbenzene (PDMS–DVB) coated fiber and determined by gas chromatography with flame ionization detector (GC-FID). To enhance the sensitivity of HS-SPME, the temperature and time of the extraction and desorption, the acidity and salt concentration of the extraction solution were optimized. Linear range of the analytes was found to be between 0.1 and 20 mg/L with regression coefficients (R 2) of 0.9998 for sorbic acid and 0.9980 for benzoic acid. Limits of detection (LOD) were 5.83 μg/L and 11.4 μg/L for sorbic and benzoic acids, respectively. Relative standard deviation (R.S.D.) for six replicate analyses within 3 days (two times/day) was found to be lower than 8.62% at three concentration levels (2, 6, 10 mg/L). Recoveries ranged from 81.20% to 108.1% for real samples. The results demonstrate the suitability of the HS-SPME technique to analyze sorbic and benzoic acids in a variety of beverages.
Keywords: HS-SPME; GC; Sorbic acid; Benzoic acid; Food preservatives; Beverages; Food analysis;

In this work, a new and simple analytical methodology for the simultaneous analysis of several quinolones (cinoxacin, oxolinic acid, nalidixic acid and flumequine) and fluoroquinolones (norfloxacin, enrofloxacin, enoxacin, ciprofloxacin and danofloxacin) in soil samples is presented. The method is based on the extraction of these analytes by an ultrasonic-assisted extraction in small columns and their subsequent quantification by HPLC using UV detection. The observed strong sorption of quinolones and fluoroquinlones to soil together their different acid–base properties made necessary an exhaustive optimisation of the extraction step. The optimum extraction procedure, based on the formation of antibiotic–Mg(II) complexes, allowed to desorb and quantitatively extract both groups of antibiotics in a single step, which was not possible by using conventional organic solvents. The proposed method was validated and the limits of detection achieved were in the low μg g−1 concentration range proving its suitability for the determination of quinolones and fluoroquinolones in soil samples at realistic environmental concentration level.
Keywords: Ultrasonic-assisted extraction; Soil; HPLC-UV; Quinolones and fluoroquinolones;

Improved method for the determination of kynurenic acid in rat plasma by column-switching HPLC with post-column fluorescence detection by Shogo Mitsuhashi; Takeshi Fukushima; Junko Kawai; Masayuki Tomiya; Tomofumi Santa; Kazuhiro Imai; Toshimasa Toyo’oka (36-43).
Kynurenic acid (KYNA), an endogenous antagonist of ionotropic glutamate and α7 nicotinic receptors, was fluorometrically determined by column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. The HPLC system consists of two octadecyl silica (ODS) columns, both of which are connected with an anion-exchange column (trapping column). Following sample injection onto the HPLC column, KYNA was separated on the first ODS column with a mobile phase of H2O/acetonitrile (95/5) containing 0.1% acetic acid. The peak fraction of KYNA was trapped on the anion-exchange column by changing the position of a six-port valve and then introduced into the second ODS column. Subsequently, KYNA was detected fluorometrically as a fluorescence complex formed with zinc ion which was pumped constantly. Instrumental limit of detection was approximately 0.16 nM, which corresponded to 8.0 fmol (per 50 μl injection, signal to noise ratio 3), and the limit of quantification was 0.53 nM (signal to noise ratio 10). Intra- and inter-day relative standard deviations were 1.1–3.9% (n  = 3) and 3.0–5.3% (n  = 3), respectively. The peak of KYNA in rat plasma was clearly detected by the proposed column-switching HPLC system after a facile pretreatment procedure. Intra- and inter-day relative mean errors were −1.6–1.4% (n  = 3) and −2.4 to −0.4% (n  = 3), respectively, with a satisfactory precision (within 5.0%). A calibration curve for the determination of KYNA showed a good linearity (r 2  > 0.999) in the range of 25–200 nM. The KYNA concentrations in the plasma of male Sprague-Dawley rats (8-week-old) were 44 ± 5.5 nM (mean ± S.E., n  = 5). In ketamine-treated rats, which are animal models of schizophrenia, the plasma KYNA concentrations were significantly increased compared with those in the control rats (p  < 0.05).
Keywords: Kynurenic acid; Column-switching HPLC; Fluorometric detection; Ketamine; Schizophrenia;

It is the first time that triphenylmethane was used as an adsorbent to preconcentrate and separate trace amount of molybdenum in water samples. The effects of different parameters, such as acidity, stirring time and various metal ions, the amounts of triphenylmethane and salicyl fluorine, etc. on the enrichment yield of molybdenum have been studied to optimize the experimental conditions. Under the optimum conditions, molybdenum can be adsorbed on the surface of microcrystalline triphenylmethane loaded with salicyl fluorone by the intermolecular action strength. The possible reaction mechanism for the enrichment of molybdenum was discussed in detail in this paper. Mo(VI) can be completely separated from Pb(II), Co(II), Cu(II), Cr(III), Ni(II), Hg(II), Zn(II), Cd(II), Fe(III) and Al(III) in the solution. The proposed method was successfully applied to the determination of trace amount of molybdenum in various water samples by spectrophotometry after preconcentration using microcrystalline triphenylmethane. The preconcentration factor is from 83 (500 ml water sample was enriched to 6.0 ml) to 166 (1000 ml water sample was enriched to 6.0 ml). The detection limit is 1.3 × 10−5  mg l−1 and the linearity is maintained in the concentration range 3.8 × 10−3 to 0.36 mg l−1 with a correlation coefficient of 0.9998. The recoveries are in the range of 93.5–104%. The relative standard deviation is 1.8–2.9%. Analytical results obtained with this novel method are very satisfactory.
Keywords: Molybdenum; Salicyl fluorone; Preconcentration; Microcrystalline triphenylmethane; Various water samples;

The fractionation and determination procedures for the speciation of 210Pb and 210Po in soil samples by Guogang Jia; Maria Belli; Senlin Liu; Umberto Sansone; Changheng Xu; Silvia Rosamilia; Xuefu Xiao; Stefania Gaudino; Ling Chen; Hetao Yang (51-58).
A method for speciation and determination of 210Pb and 210Po in soil samples was developed. The speciation was carried out by fractionating the soil samples into five fractions which are water soluble or exchangeable, bound to carbonates, bound to Fe–Mn oxides, bound to organic matter and bound to residue. After mineralisation, 10% solution of each fraction was used to spontaneously deposit polonium on a silver disk at 85–90 °C and pH 1.5, and 210Po was measured by α-spectrometry; the remain solution was used to separate lead by anion-exchange resin and purified by precipitation as PbS and PbSO4, and 210Pb was determined by a low background β-counter. The IAEA-327 reference material (soil) was studied for 210Pb and 210Po speciation. The results show that: (1) the average yields are 88.7 ± 6.4% for 210Pb and 93.8 ± 8.2% for 210Po; (2) if compared to the total 210Pb activity in the sample, 210Pb fractions are 0.95% in exchangeable form, 10.6% bound to carbonates, 14.3% bound to Fe–Mn oxides, 7.0% bound to organic matter and 67.2% bound to residue or acid soluble, and the corresponding values for 210Po are 0.17%, 0.97%, 21.0%, 0.47% and 77.4%, respectively; and (3) the obtained 210Pb concentration is in good agreement with the recommended value given by the IAEA.
Keywords: 210Pb; 210Po; Speciation; Soil;

Structural investigation on ion-selective ionophoric properties of armed 12-oxacrown-3 derivatives by Takayo Moriuchi-Kawakami; Tsukasa Yokou; Haruhisa Tsujioka; Rie Aoki; Keiichi Fujimori; Yasuhiko Shibutani (59-65).
We designed and synthesized new armed 12-oxacrown-3s bearing oxygen donor arms and forming encapsulated complexes with metal ions. The ISEs based on the single armed 12-oxacrown-3s exhibited Na+ ion selectivity, while the ISE based on the double armed 12-oxacrown-3 exhibited Li+ ion selectivity. The conformational analysis was performed on the free armed 12-oxaciown-3s and the non-encapsulated and the encapsulated armed 12-oxacrown-3 complexes using a semi-empirical method. The conformational analysis indicated that all armed 12-oxacrown-3s structurally prefer the Na+ ion rather than the Li+ ion. Further, it became apparent that the single armed 12-oxacrown-3s without a guest cation have the C 3v 12-oxaciown-3 ring and the double armed 12-oxacrown-3 without a guest cation has the bent 12-oxacrown-3 ring. The oxygens except carbonyl oxygens were directed toward the cation center in the structures of the complexes. It was clear that the ether and ester oxygens participate in the sidearm coordination.
Keywords: Computational chemistry; Conformational analysis; 12-Oxacrown-3; Sidearm; Armed crown ethers; Ion selectivity; Ion-selective electrode;

Genetic algorithm (GA) was employed to optimize the buffer system of micellar electrokinetic capillary chromatography (MEKC) for separating the active components contained in Chinese medicines. ‘SHUANGDAN’ granule, an important botanical drug in the treatment of cardiovascular diseases in China, was studied as a typical example. The optimized buffer system (25 mM borate, 29 mM phosphate and 50 mM SDS) provides the best separation with regard to resolution and analysis time. In order to validate the performance of the optimized result, different analytical parameters (e.g., precision, linearity range and recovery) of MEKC method were calculated based on the optimized separation to simultaneously determine protocatechuic aldehyde, paeonol, danshensu and salvianolic acid B contained in ‘SHUANGDAN’ granule. It was shown that GA is an effective tool for optimizing the separation of a series of active components contained in Chinese medicines by capillary electrophoresis.
Keywords: Genetic algorithm; Micellar electrokinetic capillary chromatography; Chinese medicines; Optimization;

A set of 68 active pharmaceutical substances (mainly basic, but also some neutral and acidic) was earlier used to determine the orthogonality/similarity of chromatographic systems. The orthogonality of the systems was evaluated from correlation coefficients-based weighted-average-linkage (WAL) dendrograms and color maps. To increase the throughput in assessing systems, a representative subset of substances that leads to analogous conclusions about orthogonality/similarity was selected. Both the Kennard and Stone (K&S) algorithm applied on the autoscaled principal component analysis data from the Weighted Holistic Invariant Molecular (WHIM) descriptors of the test molecules, and WAL dendrograms on retention data were used for that purpose. A subset of 10 substances was found to give similar conclusions about orthogonality and similarity of the systems examined.
Keywords: Orthogonal chromatographic systems; Kennard and Stone algorithm; Weighted-average-linkage dendrogram; Correlation coefficients color map; Weighted holistic invariant molecular descriptors;

This manuscript proposes an approach to determine three tetracyclines (tetracycline, oxytetracycline and chlortetracycline) in surface water by photochemically induced fluorescence (PIF) detection. Ternary mixtures of these tetracyclines have been simultaneously determined by application of a multivariate calibration partial least squares (PLS) method. This methodology was applied to their determination in water samples using solid phase pre-concentration (SPE) with Oasis HLB cartridges. A suppression of the PIF signal for the three tetracyclines was found using SPE, and piecewise direct standardization (PDS), direct standardization (DS) and orthogonal signal correction (OSC) were used to transfer spectra obtained after SPE to spectra registered in pure solvent. PDS performed better than DS and OCS and, therefore, it was chosen as standardization method to transfer spectra. The application of the standardization method allows the determination of the three tetracyclines at concentration levels between 3 and 10 μg l−1 in surface water samples using a training set built with standards prepared in solvent, without SPE step.
Keywords: Tetracyclines; water; Photochemically induced fluorescence; Standardization; Piecewise direct standardization (PDS); Partial least squares (PLS);

Local resolution of two-way data from multicomponent equilibria by H. Abdollahi; M.H. Sorouraddin; A.H. Naseri (94-102).
A self-modeling curve resolution (SMCR) method is proposed to calculate concentration and spectral profiles for the two-way spectral data from an equilibrium containing several chemical components. The proposed method has three main distinctive steps: (i) fixed size moving window evolving factor analysis (FSMWEFA) is used to identify the selective and zero concentration regions for a desired component, (ii) orthogonal projection resolution (OPR) is used to calculate its concentration profile and (iii) the component striping is done directly to resolve other components. The results of simulated and real polyprotic acid dissociation equilibria showed that the proposed combined method performs well even in situation when the successive stepwise equilibrium constants are close to each other. The applicability of method for resolving the triprotic acid system with rank deficiency due full spectral overlapping of two involved chemical species also is shown.
Keywords: Local resolution; Fixed size moving window evolving factor analysis (FSMWEFA); Orthogonal projection resolution (OPR);

Photochemical immobilization of protein on the inner wall of a microchannel and Its application in a glucose sensor by Hizuru Nakajima; Satomi Ishino; Hironori Masuda; Tatsuro Nakagama; Takuya Shimosaka; Katsumi Uchiyama (103-109).
A new protein immobilization technique has been developed for patterning enzymes in a specific position inside a microchannel. First, bovine serum albumin (BSA) was adsorbed onto the internal surface of a polydimethylsiloxane microchannel. The microchannel was then filled with the conjugate solution of a photoreactive cross-linker, 4-azido-2,3,5,6-tetrafluorobenzoic acid succinimidyl ester (ATFB-SE), and an enzyme, horseradish peroxidase (HRP). An irradiation by a He–Cd laser activated the azido group of the conjugates and these conjugates became covalently attached to the adsorbed BSA on the microchannel. The enzyme turnover was observed from only the HRP zone. This technique was successfully applied to the enzymatic glucose sensor. Glucose oxidase (GOD) and HRP were sequentially patterned in a single microchannel, i.e., the HRP zone was located downstream from the GOD zone. The calibration curve of a glucose standard solution was linear over the range of 0–128 μM with a correlation coefficient of 0.993. Compared to the traditional method using a 96-well microtiter plate, the present technique on the microchip shortened the reaction time from 30 min to 4.8 s, i.e., to 1/375.
Keywords: Microchip; Photochemistry; Immobilization; Patterning; Enzyme; Glucose;

In this work, the mechanochemical synthesis of a moderately agglomerated tin oxide (SnO2) powders and the subsequent preparation of semiconductor gas sensors as prototypes, were studied. Tin (II) chloride (SnCl2) powder was milled with calcium hydroxide (Ca(OH)2) and potassium carbonate, (K2CO3) powder, respectively, in a ball mill at room temperature and in an air atmosphere. Heat treatment of milled mixtures at 400 °C resulted in the formation of a tetragonal phase, confirmed by X-ray diffraction (XRD). During milling in the presence of water, a high number of hydroxide (OH) groups are formed at the surface. When SnCl2 was milled with K2CO3, no water was produced and the Fourier-transform infrared spectrum (FT-IR) of the powder has no surface hydroxyl deformations. On exposure to hydrogen sulfide (H2S) gas, the particles, prepared from anhydrous powder, have higher sensitivity than these, prepared from hydrated powder. The SnO2 thick film, prepared from anhydrous powder may be successfully applied to a H2S gas sensor.
Keywords: Mechanochemical synthesis; SnO2 powder; X-ray diffraction (XRD); Fourier transform infrared (FT-IR); SnO2 thick film; H2S gas response;

Biosensors based on highly sensitive acetylcholinesterases for enhanced carbamate insecticides detection by Bogdan Bucur; Didier Fournier; Andrei Danet; Jean-Louis Marty (115-121).
This paper presents the construction of amperometric biosensors for the highly sensitive detection of carbamate insecticides based on the inhibition of acetylcholinesterase (AChE). This enzyme was immobilised by entrapment in an optimised sol–gel matrix on TCNQ-modified screen-printed electrodes. The enzyme activity was estimated by measuring the thiocholine produced by the enzymatic hydrolysis of the acetylthiocholine using TCNQ as mediator. Wild and genetically engineered AChEs from Drosophila melanogaster (Dm) were chosen for their high sensitivity towards insecticides, which substantially improves the LOD compared with cholinesterases from other sources. The wild type and three mutant enzymes were tested against three carbamate insecticides: carbaryl, carbofuran and pirimicard. The best LOD were obtained with the Y370A mutant for carbaryl (1 × 10−8  M), the E69W mutant for pirimicarb (2 × 10−8  M) and the I161V mutant for carbofuran (8 × 10−10  M). The biosensors were applied to the analysis of two potable water samples.
Keywords: Recombinant acetylcholinesterase; Carbamate insecticide; Sol–gel immobilisation; Disposable biosensor; Screen-printed electrode;

Isoproturon was extracted selectively from environmental materials (water samples) using an immunosorbent column containing anti-isoproturon antibodies encapsulated in a silica matrix by a sol–gel process. A phosphate buffered saline (PBS) conditioned immunosorbent column was used to on-line preconcentrate 5 ml well and tap water containing 0.05 μg l−1 of isoproturon, which were desorbed with 75 μl of citric acid and determined with a solid phase competitive fluoroimmunoassay. The solid phase of the immunosensor, consisting of a sol–gel glass doped with anti-isoproturon monoclonal antibody, was placed on the flow-cell of the spectrofluorometer. Free isoproturon in solution competed with a fluorescent conjugated isoproturon and reduced the support bonded fluorescence in a concentration-dependent manner. The on-line method has a detection limit of 9.7 ng l−1, relative standard deviation of 4 and 3% for 0.05 and 0.5 μg l−1, respectively, and recoveries higher than 90% for tap and well water. For comparison the off-line extraction and clean up using a C18 cartridge is also reported.
Keywords: Sol–gel; Immunosorbent; Immunosensor; Isoproturon;

An atomic fluorescence spectrometry system for determination of total and inorganic mercury with electromagnetic induction-assisted heating on-line oxidation has been developed. Potassium peroxodisulphate was used as the oxidizing agent to decompose organomercury compounds. Depending on the temperature selected, inorganic or total mercury could be determined with the same manifold. Special accent was put on the study of the parameters influencing the on-line digestion efficiency. The tolerance to the interference of coexisting ions was carefully examined in this system. Under optimal conditions, the detection limits (3σ) were evaluated to be 2.9 ng l−1 for inorganic mercury and 2.6 ng l−1 for total mercury, respectively. The relative standard deviations for 10 replicate determinations of 1.0 μg l−1 Hg were 2.4 and 3.2% for inorganic mercury and total mercury, respectively. The proposed method was successfully applied to the determination of total and inorganic mercury in fish samples.
Keywords: Atomic fluorescence spectrometry; On-line oxidation; Inorganic mercury; Total mercury; Electromagnetic induction oven;