Analytica Chimica Acta (v.599, #1)

Contents (v-vi).

A new LC–ESI-MS/MS method to measure long-chain acylcarnitine levels in cultured cells by Olga Jáuregui; Adriana Y. Sierra; Patricia Carrasco; Esther Gratacós; Fausto G. Hegardt; Núria Casals (1-6).
The quantitative evaluation of long-chain acylcarnitines in lipid extracts from cultured cells or tissues is a prerequisite to study carnitine palmitoyltransferase (CPT) activity. There is thus a need for the accurate measurement of the concentration of long-chain acylcarnitines at the lowest concentration present in lipid extracts. Here we report a fast and reliable quantitative method based on the use of weak acid extraction and liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) to quantify acylcarnitines through hydrophilic interaction chromatography. The method was validated using isotopic dilution and the results allow the analysis of a large number of samples at low concentration levels (down to 0.35 nmol L−1 for palmitoylcarnitine) with good inter- and intra-day precision. The method was used for the quantitative study of changes in concentration of palmitoylcarnitine and other acylcarnitines in PC-12 cells over-expressing CPT1a gene. It was also used to measure CPT1 activity in mitochondria isolated from transfected cells, giving similar results to the more common radiometric method, but with higher sensitivity.
Keywords: Acylcarnitines; Carnitine palmitoyltransferase activity; Liquid chromatography–electrospray ionization tandem mass spectrometry; Hydrophilic interaction chromatography;

A large variety of potentiometric biosensors is developed using biocatalytic and bioaffinity-based biosensing schemes. However, only few of them could be applied for the biomedical analysis. The most promising are those for the detection of main products of protein metabolism, namely urea and creatinine. A novel group of potentiometric biosensors is constituted by bioaffinity-based devices that could be used for immunoassays or genoanalysis. This paper reviews the recent trends in these fields as well as discusses advantages, limitations and pitfalls of the developed biosensors. Some potentiometric biosensors useful for real biomedical analysis are reported in detail.
Keywords: Clinical analysis; Potentiometry; Biosensors; Biocatalysis; Bioaffinity;

The paper describes analytical methods developed for the production date determination of uranium-based nuclear materials by the measurement of 230Th/234U isotope ratio. An improved sample preparation method for the destructive analysis involving extraction chromatographic separation with TEVA™ resin was applied prior to the measurement by isotope dilution inductively coupled plasma sector field mass spectrometry (ICP-SFMS). The results obtained were compared with the direct, quasi-non-destructive measurement using laser ablation ICP-SFMS technique for age determination. The advantages and limitations of both methods are discussed.
Keywords: Age determination; Uranium; Inductively coupled plasma mass spectrometry; Laser ablation; Illicit trafficking; Nuclear forensics;

Multivariate calibration problems often involve the identification of a meaningful subset of variables, from a vast number of variables for better prediction of output variables. A new graph theoretic method based on partial correlations (variable interaction network—VIN) is proposed. Many well studied representative calibration datasets spanning different application domains are selected for investigating the performance. Partial least squares (PLS) regression models combined with variable selection techniques are employed for benchmarking the performance. Subsets of variables with different number of variables are retained for the final analysis after VIN selection and progressive prediction accuracies are used for comparison. VIN–PLS results show significant improvement in prediction efficiencies and variable subset optimization. Improvement of up to 45% over existing methods with significantly fewer variables is achieved using the new method. Advantages of VIN based variable selection are highlighted.
Keywords: Variable selection; Variable interaction network; Multivariate calibration; Partial correlation; Partial least squares; Multivariate statistics;

Optimization of a glucose biosensor setup based on a Ni/Al HT matrix by A. Mignani; G. Luciano; S. Lanteri; R. Leardi; E. Scavetta; D. Tonelli (36-40).
An amperometric glucose biosensor was developed using an anionic clay matrix of hydrotalcitic nature (Ni/Al–NO3 HT) as enzyme support, which was electrochemically synthesized at −0.90 V versus SCE, using a rotating disk Pt electrode to assure homogeneity of the electrodeposition suspension. The biorecognition element was glucose oxidase (GOx) immobilized on HT during the electrosynthesis, which was followed by cross-linking with glutaraldehyde vapours to avoid the enzyme release.The performances of the biosensor, in terms of sensitivity to glucose calculated from the slope of the calibration curve, are dependent on parameters related to the electrodeposition.An experimental design was applied to detect the optimal conditions of electrosynthesis in order to optimize the glucose biosensor performance. The factors taken into account were enzyme concentration and Ni/Al molar ratio. A full factorial design was performed to study linear interactions between factors and their quadratic effects and the optimal setup was evaluated by the isoresponse curves. The significant factors were enzyme concentration (linear and quadratic terms) and the interaction between enzyme concentration and Ni/Al molar ratio. Under the optimized electrodeposition conditions, the reproducibility of the biosensor fabrication was very good, being the RSD of the sensitivity about 5%.
Keywords: Experimental design; Anionic clays; Electrosynthesis; Biosensors; Glucose oxidase;

A comparison between the determination of free Pb(II) by two techniques: Absence of gradients and Nernstian equilibrium stripping and resin titration by Giancarla Alberti; Raffaela Biesuz; César Huidobro; Encarnació Companys; Jaume Puy; Josep Galceran (41-50).
Absence of gradients and Nernstian equilibrium stripping (AGNES) is an emerging electroanalytical technique designed to measure free metal ion concentration. The practical implementation of AGNES requires a critical selection of the deposition time, which can be drastically reduced if the contribution of the complexes is properly taken into account.The resin titration (RT) is a competition method based on the sorption of metal ions on a complexing resin. The competitor here considered is the resin Chelex 100 whose sorbing properties towards Pb(II) are well known. The RT is a consolidated technique especially suitable to perform an intercomparison with AGNES, due to its independent physicochemical nature.Two different ligands for Pb(II) complexation have been analyzed here: nitrilotriacetic acid (NTA) and pyridinedicarboxylic acid (PDCA). The complex PbNTA is practically inert in the diffusion layer, so, for ordinary deposition potentials, its contribution is almost negligible; however, at potentials more negative than −0.8 V vs. Ag/AgCl the complex dissociates on the electrodic surface giving rise to a second wave in techniques such as normal pulse polarography. The complex Pb–PDCA is partially labile, so that its contribution can be estimated from an expression of the lability degree of the complex. These new strategies allow us to reduce the deposition time. The free Pb(II) concentrations obtained by AGNES and by RT are in full agreement for both systems here considered. The main advantage of the use of AGNES in these systems lies in the reduction of the time of the experiment, while RT can be applied to non-amalgamating elements and offers the possibility of simultaneous determinations.
Keywords: Free lead(II) concentration; Stripping analysis; Lability degree; Chelex 100; Speciation; Absence of gradients and Nernstian equilibrium stripping;

Assembly of polyoxometalates on carbon nanotubes paste electrode and its catalytic behaviors by Jianying Qu; Xiangqin Zou; Baifeng Liu; Shaojun Dong (51-57).
Carbon nanotubes paste (CNTP) electrode was prepared with multi-walled carbon nanotubes and methyl silicone oil. Polyoxometalates (POMs) were assembled on the electrode surface with different methods, and investigated by cyclic voltammetry and Raman spectroscopy. Experiments showed that POMs/CNTP electrode prepared by direct method had better performance. K6P2Mo18O62·14H2O (P2Mo18) assembled CNTP electrode (P2Mo18/CNTP) electrode possessed good reversibility and could catalyze the reduction of bromate and iodate in 0.1 M H2SO4 solution. Further, the multilayer films of P2Mo18 assembled CNTP electrodes were fabricated by layer-by-layer technique, which showed higher electrocatalytic activities. All these POMs assembled CNTP electrodes prepared exhibited good stability.
Keywords: Carbon nanotube paste electrode; Polyoxometalates; Assembly; Catalyze;

Determination of humic substances in natural waters by cathodic stripping voltammetry of their complexes with iron by Luis M. Laglera; Gianluca Battaglia; Constant M.G. van den Berg (58-66).
A new voltammetric method is presented for the measurement of humic substances (HS) in natural waters. The method is based on catalytic cathodic stripping voltammetry (CSV) and makes use of adsorptive properties of iron–HS complexes on the mercury drop electrode at natural pH. A fulvic acid standard (IHSS) was used to confirm the voltammetric response (peak potential and sensitivity) for the HS for natural water samples. Optimized conditions included the linear-sweep mode, deposition at −0.1 V, pH buffered at 8 and a scan rate of 50 mV s−1. At a deposition time of 240 s in the presence of 10 nM iron and 30 mM bromate, the detection limit was 5 μg L−1  HS in seawater, which could be lowered further by an increase in the bromate concentration, or in the adsorption time. The method was used to determine HS in the Irish Sea which were found to occur at levels between 60 and 600 μg L−1. The new method is sufficiently sensitive to detect the low HS content in oceanic samples and has implications to the study of iron speciation.
Keywords: Fulvic acid; Humic substances; Seawater; Natural waters; Cathodic stripping voltammetry; Iron;

Complexing capacity profiles of naturally occurring ligands in Tempranillo wines for Cu and Zn by I. Esparza; C. Santamaría; J.M. García-Mina; J.M. Fernández (67-75).
Complexing capacity of naturally occurring ligands in Vitis vinifera (Tempranillo variety) wines has been studied with respect to two target metals (Cu and Zn) by differential pulse anodic stripping voltammetry (DPASV). Eight commercial wines of two certified brands of origin (CBO) and a young wine along its vinification process were monitored. Conditional stability constants and total complexing ligand(s) concentration(s) have been calculated for both metals. Discussion of the particular electrochemical responses for Cu and Zn for all samples is presented. A follow-up of the Cu stripping response allowed differentiating a commercial wine from one under processing related to the cupric casse phenomenon. Interaction of Cu with two molecular forms of cyanidin has been theoretically modeled at natural wine pH.
Keywords: Complexation; Wine; Vinification; Cu; Zn; Cupric casse; Differential pulse anodic stripping voltammetry;

In-drop derivatisation single-drop microextraction approach can constitute, to a certain degree, a low-cost reasonable alternative to the well-known on-fibre solid-phase microextraction. The headspace mode integrates extraction, preconcentration and derivatisation into a single step from the headspace of a sample. In this study, two low-molecular-weight aldehydes are derivatised in a hanging drop containing 2,4,6-trichlorophenylhydrazine, in a headspace single-drop microextraction configuration system. The single organic drop, dispersed in gas phase, is well covered in this study as a locale of the main reaction. The measurement of diffusion and kinetic parameters and their relationship were designed to reveal, for the first time, inherent mechanistic aspects in such an analytical system. The two-film theory of mass transfer is used to discuss the mechanism along with the calculation of characteristic times and specific rates of absorption. All these, together with certain experimental data may ascertain whether the overall process is reaction rate dependent or limited by mass transfer in the gas phase, at the air–water and air–organic interface or in the organic phase. The descriptors of mass transfer and chemical reaction in a single drop are critically reviewed and reconsidered and the practical aspects for the analysis of volatile organic compounds are highlighted. Relative standard deviations for both aldehydes were 3.4% (n  = 5) and 4.9% (n  = 5) for 1 μM of hexanal and 0.3 μM of formaldehyde, respectively. Detection limits for aqueous samples were 0.1 and 0.03 μM for formaldehyde and hexanal, respectively.
Keywords: Headspace single-drop microextraction; In-drop derivatisation; Theoretical analysis; Aldehydes;

An alternative sorptive microextraction method for the determination of five halogenated anisoles in water and wine matrices is proposed. Analytes were concentrated in an inexpensive and disposable piece of bulk polydimethylsiloxane (PDMS), desorbed with a small volume of organic solvent, and determined by gas chromatography with electron–capture detection (GC–ECD) or tandem mass spectrometry (GC–MS/MS). The influence of several factors on the efficiency of extraction and desorption steps was investigated in detail and the observed behaviour justified on the basis of thermodynamics and kinetics of the solid-phase microextraction technique. Under optimised conditions, analytes were first extracted in the headspace (HS) mode, at room temperature, for 2.5 h and then desorbed with 1 mL of n-pentane. This extract was further evaporated to 50 μL. The overall extraction yield of the procedure ranged from 40 to 55% and the limits of quantification remained between 0.5 and 20 ng L−1, depending on the compound considered and the detection technique. Precision and linearity of the method were excellent for all species with both GC–ECD and GC–MS/MS detection. Matrix effects were evaluated with different water and wine samples; moreover, the suitability of the PDMS sorbent for storage of analytes, under different conditions, was demonstrated.
Keywords: Anisoles; Polydimethylsiloxane; Sorptive extraction; Gas chromatography;

A new approach has been developed for the monitoring of linear alkylbenzene sulfonates (LAS) (C10–C13) in sewage sludge. It is based on microwave-assisted extraction (MAE) prior to high performance liquid chromatography coupled with diode array (HPLC-DAD) and fluorescence (HPLC-FL) detectors. Column Zorbax XDB-C8, 150 mm × 4.6 mm i.d., 5 μm particle size was used as well as acetonitrile–water containing 0.1 M NaClO4 (65:35) and isocratic elution. Compounds were isocratically eluted over 6 min runtime at a flow rate of 1 mL min−1. Since polar interferences are eluted between 0 and 2 min, they do not interfere in the analysis. The column was thermostated at a temperature of 25 °C. For the determination of LAS, DAD-UV (λ ex  = 225 nm) and fluorescence (λ ex  = 225 nm, λ em  = 295 nm) detectors were employed.The main factors affecting the extraction efficiency (extractant volume, microwave power and irradiation time) were optimized using a compost sludge. The detection limit for total LAS in the sludge was <5 mg kg−1. The extraction of C10–C13 homologues is carried out by using an extraction time of 10 min and 5 mL of methanol; whereas Soxhlet needs a 12 h extraction. The method did not require clean-up or preconcentration steps. Concentration levels of LAS were between 13037 mg kg−1 for digested sludge and 2492 mg kg−1 for compost sludge.
Keywords: Sewage sludge; Alkylbenzene sulfonates; Microwave-assisted extraction; Liquid chromatography;

The liquid chromatography–electrospray ionization-tandem multi-stage mass spectrometry (LC–ESI-MS n ) method was developed for the analyses and characterization of steroidal saponins in plant extract from the rhizome of Dioscorea nipponica Makino. The HPLC experiments were performed by means of a reversed-phase C18 column and a binary mobile phase system consisting of water and acetonitrile under gradient elution conditions. Pseudoprotodioscin, methyl protodioscin and dioscin were identified by comparing the retention times, UV spectra and the fragmentation properties of [M  − H] ions with the authentic standards. Four groups of steroidal saponin isomers possessed the [M  − H] ions at m/z 1063, 1045, 901 and 1047, respectively, were observed during the LC–ESI(−)-MS analysis, and three groups of them except the pair of isomers with the [M  − H] ions at m/z 1047 could be differentiated by LC–ESI(−)-MS3. Furthermore, the ESI-MS n fragmentation behaviors of the [M  + Li]+ ions of pseudoprotodioscin and methyl protodioscin have been investigated, and the observed information helped the structural elucidation of the more abundant isomer with the [M  − H] ion at m/z 1047. As the result, a special sugar sequence of the saccharide chains was observed that not glucose but rhamnose might be connected with the hydroxyl group at C-3 position of the steroidal aglycone.
Keywords: Dioscorea nipponica Makino; Steroidal saponin; Electrospray ionization tandem mass spectrometry; Structural differentiation;

Paint cross-sections have been analysed using the attenuated total reflection technique combined with FTIR mapping microspectroscopy in order to characterise the nature of the compounds present and map their localisation in the stratigraphy. The study reveals the possibilities offered by micro-ATR devices for obtaining informations about the organic substances employed in painting techniques and in particular their distribution in the different layers, showing a real improvement over traditional analytical investigations in use for the detection of organic substances. Limitations, such as the contamination of the embedding resin and the typical spectral resolution (20 μm) are presented and alternative methods were proposed to obtain better results. In particular, the use of an infrared transparent salt (KBr) as embedding material for the cross-sections is evaluated and seems to be very promising. Furthermore, ATR mapping represent a useful non-destructive analytical technique complementary to others molecular and elemental analyses to be performed afterwards such as SEM-EDX.
Keywords: Attenuated Total Reflection Mapping; Fourier Transform Infrared mcrospectroscopic imaging; Conservation studies; Painting materials analysis;

We demonstrate the resonance wavelength-dependent signal of colloidal gold nanoparticles adsorbed to a planar gold surface in surface plasmon resonance (SPR)-based detection. Experimental measurements of the SPR signal as a function of particle surface coverage are presented for three different resonance wavelengths. The SPR signal due to the colloidal gold nanoparticles varies across the resonance wavelengths of 650 nm, 770 nm, and 920 nm. The experimental SPR curves show good agreement with the results of a Lorentz absorbance model at the lower particle surface coverages investigated. The results demonstrate an almost twofold signal difference for a subset of the experimental conditions explored.
Keywords: Surface plasmon resonance; Gold colloid; Nanoparticle; Signal amplification;

Polymerized Nile Blue derivatives for plasticizer-free fluorescent ion optode microsphere sensors by Wittaya Ngeontae; Chao Xu; Nan Ye; Katarzyna Wygladacz; Wanlapa Aeungmaitrepirom; Thawatchai Tuntulani; Eric Bakker (124-133).
Lipophilic H+-selective fluorophores such as Nile Blue derivatives are widely used in ISE-based pH sensors and bulk optodes, and are commonly dissolved in a plasticized matrix such as PVC. Unfortunately, leaching of the active sensing ingredients and plasticizer from the matrix dictates the lifetime of the sensors and hampers their applications in vivo, especially with miniaturized particle based sensors. We find that classical copolymerization of Nile Blue derivatives containing an acrylic side group gives rise to multiple reaction products with different spectral and H+-binding properties, making this approach unsuitable for the development of reliable sensor materials. This limitation was overcome by grafting Nile Blue to a self-plasticized poly(n-butyl acrylate) matrix via an urea or amide linkage between the Nile Blue base structure and the polymer. Optode leaching experiments into methanol confirmed the successful covalent attachment of the two chromoionophores to the polymer matrix. Both polymerized Nile Blue derivatives have satisfactory pH response and appropriate optical properties that are suitable for use in ion-selective electrodes and optodes. Plasticizer-free Na+-selective microsphere sensors using the polymerized chromoionophores were fabricated under mild conditions with an in-house sonic microparticle generator for the measurement of sodium activities at physiological pH. The measuring range for sodium was found as 10−1–10−4  M and 1–10−3  M, for Nile Blue derivatives linked via urea and amide functionalities, respectively, at physiological pH. The observed ion-exchange constants of the plasticizer-free microsphere were log  K exch  = −5.6 and log  K exch  = −6.5 for the same two systems, respectively. Compared with earlier Na+-selective bulk optodes, the fabricated optical sensing microbeads reported here have agreeable selectivity patterns, reasonably fast response times, and more appropriate measuring ranges for determination of Na+ activity at physiological pH in undiluted blood samples.
Keywords: Chromoionophore; Fluoroionophore; Nile Blue; Copolymerization; Plasticizer free; Fluorescent sensors; Microsphere ion sensor;

A functionalized gold nanoparticles and Rhodamine 6G based fluorescent sensor for high sensitive and selective detection of mercury(II) in environmental water samples by Jinlong Chen; AiFang Zheng; AiHong Chen; Yingchun Gao; Chiyang He; Xiaoming Kai; Genhua Wu; Youcun Chen (134-142).
A gold-nanoparticles (Au NPs)–Rhodamine 6G (Rh6G) based fluorescent sensor for detecting Hg (II) in aqueous solution has been developed. Water-soluble and monodisperse gold nanoparticles (Au NPs) has been prepared facilely and further modified with thioglycolic acid (TGA). Free Rh6G dye was strongly fluorescent in bulk solution. The sensor system composing of Rh6G and Au NPs fluoresce weakly as result of fluorescence resonance energy transfer (FRET) and collision. The fluorescence of Rh6G and Au NPs based sensor was gradually recovered due to Rh6G units departed from the surface of functionalized Au NPs in the presence of Hg(II). Based on the modulation of fluorescence quenching efficiency of Rh6G–Au NPs by Hg(II) at pH 9.0 of teraborate buffer solution, a simple, rapid, reliable and specific turn-on fluorescent assay for Hg(II) was proposed. Under the optimum conditions, the fluorescence intensity of sensor is proportional to the concentration of Hg(II). The calibration graphs are linear over the range of 5.0 × 10−10 to 3.55 × 10−8  mol L−1, and the corresponding limit of detection (LOD) is low as 6.0 × 10−11  mol L−1. The relative standard deviation of 10 replicate measurements is 1.5% for 2.0 × 10−9  mol L−1 Hg(II). In comparison with conventional fluorimetric methods for detection of mercury ion, the present nanosensor endowed with higher sensitivity and selectivity for Hg(II) in aqueous solution. Mercury(II) of real environmental water samples was determined by our proposed method with satisfactory results that were obtained by atomic absorption spectroscopy (AAS).
Keywords: Gold particles; Rhodamine 6G; Fluorescence sensor; Mercury(II);

Determination of nateglinide in human plasma by high-performance liquid chromatography with pre-column derivatization using a coumarin-type fluorescent reagent by Danai Malli; Evagelos Gikas; Andreas Vavagiannis; Michael Kazanis; Konstantinos Daniilides; Dimitra Gennimata; Irene Panderi (143-150).
A sensitive and selective high-performance liquid chromatographic method has been developed and validated for the determination of nateglinide in human plasma. Nateglinide and the internal standard, undecylenic acid, were extracted from plasma by liquid–liquid extraction using a mixture of ethyl acetate–diethyl ether, 50:50 (v/v). Pre-column derivatization reaction was performed using a coumarin-type fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The derivatization proceeded in acetone in the presence of potassium carbonate and catalyzed by 18-crown-6 ether. The fluorescent derivatives were separated under isocratic conditions on a Hypersil BDS-C8 analytical column (250.0 mm × 2.1 mm i.d., particle size 5 μm) with a mobile phase that consisted of 65% acetonitrile in water and pumped at a flow rate of 0.50 mL min−1. The excitation and emission wavelengths were set at 345 and 435 nm, respectively. The assay was linear over a concentration range of 0.05–16.00 μg mL−1 for nateglinide with a limit of quantitation of 0.05 μg mL−1. Quality control samples (0.05, 4.50 and 16.00 μg mL−1) in five replicates from five different runs of analysis demonstrated intra-assay precision (%coefficient of variation <6.8%), inter-assay precision (%coefficient of variation <1.6%) and an overall accuracy (%relative error) less than −3.4%. The method can be used to quantify nateglinide in human plasma covering a variety of pharmacokinetic or bioequivalence studies.
Keywords: Nateglinide; Undecylenic acid; Coumarin; Pre-column fluorescence derivatization; High-performance liquid chromatography; Human plasma;

The efficiency of capillary columns in programmed temperature analysis can be evaluated by calculation of the separation number (“Trennzahl”). A procedure for the prediction of this parameter at various initial temperatures, carrier gas pressures and heating rates, by using as the starting data the retention times and the peak widths obtained in some isobaric and isothermal runs is described. An equation is proposed that permits to obtain the values of the peak width at half height in any isothermal and linearly programmed temperature gas chromatographic run and therefore to calculate the separation number value. The effect on this parameter of the column polarity was investigated by using polar and non-polar compounds (n-alkanes and 1-alcohols).
Keywords: Capillary columns; Column efficiency; Gas chromatography; Separation number; Temperature programming; Peak width;