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Analytica Chimica Acta (v.650, #2)

Editorial Board (pp. i).

Overview on modern approaches to speed up protein identification workflows relying on enzymatic cleavage and mass spectrometry-based techniques by J.L. Capelo; R. Carreira; M. Diniz; L. Fernandes; M. Galesio; C. Lodeiro; H.M. Santos; G. Vale (pp. 151-159).

Recent tools addressed to accelerate the different steps of the sample treatment for protein identification in modern workflows are reviewed and critically commented in this manuscript. Heating, microspin columns, ultrasonic energy, high pressure, infrared energy, microwave energy, alternating electric fields and microreactors are outlined as useful tools that can be used to accelerate all or some of the following steps for in-gel or in-liquid based approaches for protein identification: (i) protein dissolution/denaturation, (ii) protein reduction, (iii) protein alkylation and (iv) protein digestion. The advantages and drawbacks, along with the main differences among the different tools are also commented. Future prospects for hyphenation of methods are also discussed. Researchers are informed also in this work regarding the main problems to be found when implementing any of the above mentioned methods.

Keywords: Proteomics; Protein identification; Mass spectrometry; Fast methods

Simultaneous determination of psoralen and isopsoralen in plasma and Chinese medicine Xian Ling Gu Bao capsule by using HPLC-DAD coupled with alternating trilinear decomposition algorithm by Li-Qun Ouyang; Hai-Long Wu; Jin-Fang Nie; Yan Zhang; Hong-Yan Zou; Hai-Yan Fu; Ru-Qin Yu (pp. 160-166).

A method using high performance liquid chromatography with photodiode-array detection (HPLC-DAD) coupled with alternating trilinear decomposition (ATLD) algorithm was proposed for simultaneous determination of psoralen and isopsoralen in plasma and Chinese medicine “Xian Ling Gu Bao” capsule (XLGBC). In this paper, the application of ATLD algorithm into traditional chromatographic method can handle this problem that the chromatographic and spectral peaks are heavily overlapped among the analytes and even between the analytes and interferences from the background matrices. A simple improvement of chromatographic condition like mobile phase is not enough to realize effective separation for the two isomeric compounds, especially in the presence of interferences. However, the ATLD algorithm utilized “mathematical separation” instead of partial “physical or chemical separation” to directly determine the spectral profiles of the analytes of interests in complex system. The satisfactory quantification results have been gained with simple mobile phase. In the analysis of real Chinese medicine samples, the accuracy of the concentrations which were obtained by ATLD was also validated by HPLC–MS method.

Keywords: HPLC-DAD; Alternating trilinear decomposition; Psoralen; Isopsoralen; Plasma; Chinese medicine

Novel chemometric strategy based on the application of artificial neural networks to crossed mixture design for the improvement of recombinant protein production in continuous culture by Caroline Didier; Guillermina Forno; Marina Etcheverrigaray; Ricardo Kratje; Héctor Goicoechea (pp. 167-174).

The optimal blends of six compounds that should be present in culture media used in recombinant protein production were determined by means of artificial neural networks (ANN) coupled with crossed mixture experimental design. This combination constitutes a novel approach to develop a medium for cultivating genetically engineered mammalian cells. The compounds were collected in two mixtures of three elements each, and the experimental space was determined by a crossed mixture design. Empirical data from 51 experimental units were used in a multiresponse analysis to train artificial neural networks which satisfy different requirements, in order to define two new culture media (Medium 1 and Medium 2) to be used in a continuous biopharmaceutical production process. These media were tested in a bioreactor to produce a recombinant protein in CHO cells. Remarkably, for both predicted media all responses satisfied the predefined goals pursued during the analysis, except in the case of the specific growth rate ( μ) observed for Medium 1. ANN analysis proved to be a suitable methodology to be used when dealing with complex experimental designs, as frequently occurs in the optimization of production processes in the biotechnology area. The present work is a new example of the use of ANN for the resolution of a complex, real life system, successfully employed in the context of a biopharmaceutical production process.

Keywords: Artificial neural networks; Crossed mixture experimental design; Culture medium formulation

Adsorption of s-triazines onto polybenzimidazole: A quantitative structure–property relationship investigation by Angelo Antonio D’Archivio; Angela Incani; Pietro Mazzeo; Fabrizio Ruggieri (pp. 175-182).

The adsorption of 25 symmetric triazines (s-triazines) on polybenzimidazole (PBI) beads is investigated under equilibrium (batch) conditions. The observed adsorption isotherms of the selected compounds are accurately described by the Freundlich model, while the agreement between the Langmuir model and the experimental data is moderately worse, which seems to reflect the heterogeneous meso- and micro-porosity of PBI and polydispersion in the interaction mechanism. Methylthio- and methoxytriazines exhibit a greater adsorption tendency as compared with chlorotriazines, moreover, progressive dealkylation of amino groups results in a progressive increase of triazine uptake on PBI. Based on these evidences, the adsorption mechanism seems to be governed by a combination of π–π and hydrogen-bonding interactions. Genetic algorithm (GA) variable selection and multilinear regression (MLR) are combined in order to describe the effect of triazine structure on the extraction performance of PBI according to the quantitative structure–property relationship (QSPR) method. q_max, the amount of triazine adsorbed per weight unit of PBI assuming homogeneous monolayer (Langmuir) mechanism, exhibits a great variability within the set of investigated triazines and is the quantity here modelled by QSPR. On the other hand, the Freundlich constant, K_F, which expresses the adsorption efficiency under multilayer heterogeneous conditions, even if markedly increases passing from chloro- to methylthio- or methoxytriazines, is less noticeably affected by the fine details of the adsorbate structure, as the number or nature of alkyl fragments bound to the amino groups. To quantitatively relate q_max with the triazine structure GA-MLR analysis is performed on the set of 1664 theoretical molecular descriptors provided by the software Dragon. Finally, a four-dimensional QSPR model is selected based on leave-one-out cross-validation and its prediction ability is further tested on four representative triazines excluded from model calibration. The four descriptors selected by GA-MLR, all belonging to the class of three-dimensional GETAWAY (GEometry, Topology, and Atom-Weights AssemblY) descriptors, adequately represent the structural factors influencing the affinity of triazines to PBI in the batch extraction process.

Keywords: Triazines; Polybenzimidazole; Adsorption isotherms; Quantitative structure–property relationship; Molecular descriptors

Monitoring storage time and quality attribute of egg based on electronic nose by Wang Yongwei; Jun Wang; Bo Zhou; Qiujun Lu (pp. 183-188).

The objective of this study was to investigate the potential of an electronic nose (E-nose) technique for monitoring egg storage time and quality attributes. An electronic nose was used to distinguish eggs under cool and room-temperature storage by means of principal component analysis (PCA), linear discriminant analysis (LDA), BP neural network (BPNN) and the combination of a genetic algorithm and BP neural network (GANN). Results showed that the E-nose could distinguish eggs of different storage time under cool and room-temperature storage by LDA, PCA, BPNN and GANN; better prediction values were obtained by GANN than by BPNN. Relationships were established between the E-nose signal and egg quality indices (Haugh unit and yolk factor) by quadratic polynomial step regression (QPSR). The prediction models for Haugh unit and yolk factor indicated a good prediction performance. The Haugh unit model had a standard error of prediction of 3.74 and correlation coefficient 0.91; the yolk factor model had a 0.02 SEP and 0.93 correlation coefficient between predicted and measured values respectively.

Keywords: Egg; Electronic nose; Quality attribute; Storage time; Neural network

A thin-layer contactless conductivity cell for detection in flowing liquids by J. Míka; F. Opekar; P. Coufal; K. Štulík (pp. 189-194).

The design is described of a thin-layer contactless conductivity detector suitable for liquid chromatography and flow-injection analysis. Its principal analytical parameters have been determined using a potassium chloride solution: the linear dynamic range extends from 7.5×10⁻⁶ to 1.5×10⁻²Sm⁻¹, corresponding to the KCl concentration range from 0.5 to 1000μM, the limit of detection equals 3.5×10⁻⁶Sm⁻¹ (0.2μM KCl), the detection repeatability, expressed in terms of the relative standard deviation, amounts to 1.13% and the detection volume is 0.6μL. The detector was applied to detection of ionic compounds, benzoic, lactic and octanesulfonic acids, and sodium capronate, after their separation by liquid chromatography in a Biospher PSI 100C 18 columns using a 60% aqueous acetonitrile mobile phase. The frequency characteristics of the detector are reasonably theoretically described on the basis of a simple model which is commonly used in the field of contactless impedance detectors.

Keywords: Electrochemical detector; Contactless conductivity detection; Thin-layer detector; Analysis in flowing liquids; Liquid chromatography

Use of a bisphenol-A imprinted polymer as a selective sorbent for the determination of phenols and phenoxyacids in honey by liquid chromatography with diode array and tandem mass spectrometric detection by E. Herrero-Hernández; R. Carabias-Martínez; E. Rodríguez-Gonzalo (pp. 195-201).

An extraction-preconcentration procedure based on the use of a molecularly imprinted polymer (MIP) as selective sorbent has been developed for the determination of several phenolic compounds (bisphenol-A, bisphenol-F and 4-nitrophenol) and phenoxyacid herbicides (2,4-D, 2,4,5-T and 2,4,5-TP) in honey samples. Liquid chromatography with diode array detection (LC–DAD) and electrospray ionisation-ion trap mass spectrometry (LC–IT-MS) were used for the separation, identification and quantification of these analytes.The molecularly imprinted polymer was obtained by precipitation polymerisation with bisphenol-A (BPA) as template and 4-vinylpyridine as the functional monomer. The behaviour of this sorbent was compared with those of other materials frequently used in SPE. The selectivity of the BPA–MIP for the target analytes was tested in samples containing other pesticides in common use. The recoveries achieved for all six compounds were in the 81–96% range.By applying the proposed procedure prior to LC–IT-MS, the limits of detection achieved in commercial honey samples were in the 0.1–3.8ngg⁻¹ range, with relative standard deviations of 12–24%.

Keywords: Molecularly imprinted polymer; Bisphenol-A; Phenols; Phenoxyacids; Molecularly imprinted solid phase extraction; Honey

Application of solid-phase microextraction for the determination of organophosphorus pesticides in textiles by gas chromatography with mass spectrometry by Fang Zhu; Wenhong Ruan; Minheng He; Feng Zeng; Tiangang Luan; Yexiang Tong; Tongbu Lu; Gangfeng Ouyang (pp. 202-206).

A method based on solid-phase microextraction (SPME) and gas chromatography with mass spectrometry (GC/MS) for the determination of 18 organophosphorus pesticides (OPPs) in textiles is described. Commercially available SPME fibers, 100μm PDMS and 85μm PA, were compared and 85μm PA exhibited better performance to the OPPs. Various parameters affecting SPME, including extraction and desorption time, extraction temperature, salinity and pH, were studied. The optimized conditions were: 35min extraction at 25°C, 5% NaSO₄ content, pH 7.0, and 3.5min desorption in GC injector port at 250°C. The linear ranges of the SPME-GC/MS method were 0.1–500μgL⁻¹ for most of the OPPs. The limits of detection (LODs) ranged from 0.01μgL⁻¹ (for bromophos-ethyl) to 55μgL⁻¹ (for azinphos-methyl) and the RSDs were between 0.66% and 9.22%. The optimized method was then used to analyze 18 OPPs in textile sample, and the determined recoveries were ranged from 76.7% to 126.8%. Moreover, the distribution coefficients of the OPPs between 85μm PA fiber and simulative sweat solution ( K_pa/s) were determined. The determined K_pa/s of the OPPs correlated well with their octanol–water partition coefficients ( r=0.764 and 0.678) and water solubility ( r=−0.892 and −0.863).

Keywords: Organophosphorus pesticides; Solid-phase microextraction; Gas chromatography–mass spectrometry; Distribution coefficient; Textiles

Determination of benzimidazolic fungicides in fruits and vegetables by supramolecular solvent-based microextraction/liquid chromatography/fluorescence detection by Antonia Moral; María Dolores Sicilia; Soledad Rubio (pp. 207-213).

A supramolecular solvent consisting of vesicles, made up of equimolecular amounts of decanoic acid (DeA) and tetrabutylammonium decanoate (Bu₄NDe), dispersed in a continuous aqueous phase, is proposed for the extraction of benzimidazolic fungicides (BFs) from fruits and vegetables. Carbendazim (CB), thiabendazole (TB) and fuberidazole (FB) were extracted in a single step and no clean-up or concentration of extracts was needed. The high extraction efficiency obtained for BFs was a result of the different types of interactions provided by the supramolecular solvent (e.g. hydrophobic and hydrogen bonds) and the high number of solubilisation sites it contains. Besides simple and efficient, the proposed extraction approach was rapid, low-cost, environment friendly and it was implemented using conventional lab equipments. The target analytes were determined in the supramolecular extract by LC/fluorescence detection. They were separated in a Kromasil C₁₈ (5μm, 150mm×4.6mm) column using isocratic elution [mobile phase: 60:40 (v/v) 50mM phosphate buffer (pH 4)/methanol] and quantified at 286/320nm (CB) and 300/350nm (TB and FB) excitation/emission wavelengths, respectively. Quantitation limits provided by the supramolecular solvent-based microextraction (SUSME)/LC/fluorescence detection proposed method for the determination of CB, TB and FB in fruits and vegetables were 14.0, 1.3 and 0.03μgkg⁻¹, respectively, values far below the current maximum residue levels (MRLs) established by the European Union, i.e. 100–2000μgkg⁻¹ for CB, 50–5000μgkg⁻¹ for TB and 50μgkg⁻¹ for FB. The precision of the method, expressed as relative standard deviation, for inter-day measurements ( n=13) was 3.3% for CB (50μgkg⁻¹), 3.5% for TB (10μgkg⁻¹) and 2.8% for FB (0.5μgkg⁻¹) and recoveries for fruits (oranges, tangerines, lemons, limes, grapefruits, apples, pears and bananas) and vegetables (potatoes and lettuces) fortified at the μgkg⁻¹ level were in the interval 93–102%.

Keywords: Supramolecular solvent; Vesicles; Liquid chromatography; Benzimidazolic fungicides; Fruits; Vegetables

A quantitative model to evaluate the ion-enrichment and ion-depletion effect at microchannel–nanochannel junctions by Guanbin Li; Shili Wang; Chang Kyu Byun; Xiayan Wang; Shaorong Liu (pp. 214-220).

In a nanometer-scale fluidic channel (nanochannel), coions are depleted while counterions are concentrated due to the electric double layer (EDL) overlap. When an electric field is applied across the nanochannel, ions are enriched at one end and depleted at the other end of the nanochannel. This phenomenon is termed the ion-enrichment and ion-epletion (IEID) effect. In this paper, we develop a theoretical model to evaluate this effect. The model takes into accounts not only the biased electrophoretic migrations but also the net charge transportation caused by electroosmotic flow. In addition, we consider the conductance change inside the nanochannel in assessing the electric field strength across it. We employ our recently developed protocol to measure these values. We establish a protocol to measure/quantitate the IEID effect. Finally, we compare the calculated results with the experimentally measured data and show good agreements between them.

Keywords: Microchannel–nanochannel junctions; Model; Ion enrichment and ion depletion; Electric double layers; Zeta potential

Gas chromatography–mass spectrometric analysis of bonded long chain fatty acids in a single zebrafish egg by ultrasound-assisted one-step transmethylation and extraction by Jianjian Li; Yingxia Yue; Tingting Li; Xuejiao Hu; Hongying Zhong (pp. 221-226).

Changes in the level of lipid composition in a single zebrafish egg have been involved in biological responses to chemical exposures. In this paper, an one-step transmethylation of lipids and simultaneous extraction of resultant fatty acid methyl esters followed by gas chromatography–mass spectrometric analysis was developed for the identification of bonded long chain fatty acids in a single zebrafish egg. The efficiency of transmethylation under different experimental conditions has been investigated. Dried egg homogenates were directly mixed with either 0.5M NaOH or 1% H₂SO₄ or 4% HCl solution of methanol and then an n-hexane solution was added on the top. Ultrasonication of this immiscible liquid–liquid–solid system produces high velocity impacts between solid particles and liquid phases and thus promotes mass transfer among phases. It was demonstrated that ultrasound irradiation has strong effect on the alkaline-catalyzed transmethylation of lipids but cannot significantly change the acid-catalyzed transmethylation of lipids. With the aid of ultrasonication, transmethylation can be combined with simultaneous extraction of the resultant fatty acid methyl esters into n-hexane phase. This approach simplifies the sample preparation procedure, shortens the reaction time but improves the efficiency of the transmethylation of lipids and reduces sample losses, especially for small size samples. It has been applied to determine bonded fatty acids in a single zebrafish egg. In total, 28 fatty acids from a single zebrafish egg have been identified reproducibly.

Keywords: Ultrasound; Transmethylation; Bonded long chain fatty acids; Gas chromatography–mass spectrometric analysis

Purity determination and uncertainty evaluation of theophylline by mass balance method, high performance liquid chromatography and differential scanning calorimetry by Kang Ma; Haifeng Wang; Min Zhao; Jinjing Xing (pp. 227-233).

Purity determination of pure organic substance is essential for the establishment of traceability to SI units. A mass balance method was employed to determine the purity of theophylline certified reference materials (CRM), compared with high performance liquid chromatography (HPLC) and differential scanning calorimetry (DSC). In the approach of the mass balance, the impurities were identified by ion trap time-of-flight mass spectrometer (IT-TOF-MS) and quantified by HPLC. The purity of theophylline CRM determined by mass balance method was 99.82% with an extended uncertainty of 0.1% ( k=2). The uncertainty evaluation of purity demonstrated that the accuracy of the mass balance method is better than that of HPLC and DSC. It indicated that the mass balance is suitable for the CRM and pharmaceutical standards.

Keywords: Mass balance; Uncertainty; Purity; High performance liquid chromatography; Differential scanning calorimetry; Theophylline; Certified reference materials

Metal–metallothioneins like proteins investigation by heteroatom-tagged proteomics in two different snails as possible sentinel organisms of metal contamination in freshwater ecosystems by Heloisa França Maltez; Margarita Villanueva Tagle; Maria del Rosario Fernández de la Campa; Alfredo Sanz-Medel (pp. 234-240).

Metal speciation analysis in MLPs was carried out in two snails, Marisa cornuarietis and Pomacea bridgesi, in order to investigate them as possible sentinel organisms of heavy metal contamination. To carry out this study snails born in a non-contaminated environment were divided into two groups: a control group and a contaminated one with cadmium administered for 40 days. Subsequently, we investigated the speciation of the induced MLPs in exposed animals in relation to controls. In order to obtain the MLP fraction, cytosols from both snail species where subjected to size-exclusion fractionation, monitoring on-line the metal signal (Cd, Cu and Zn) by ICP-MS while protein elution was followed by on-line UV detection. MLP fraction was then separated by anion-exchange (AE)-FPLC using optimal chromatographic conditions for the separation of the different MLP isoforms in both snail species. Specific detection of separated metalloforms was carried out again by the hyphenation of the AE chromatographic system with ICP-MS. The determination of the amount of metal bound to MLPs was carried out by post-column isotope dilution analysis ICP-MS, finding that the snail M. cornuarietis accumulated higher concentrations of cadmium than P. bridgesi. Thus this first snail could therefore be a better candidate sentinel organism of pollution in natural waters. Identification and characterization of the isoforms separated in M. cornuarietis was carried out for the entire or intact isoforms by MALDI-TOF and then conventional triptic digestion was also carried out to identify the nature of the formed peptides. The presence identification of a MLP isoform of relatively low molecular weight in M. cornuarietis is reported.

Keywords: Metallothionein like proteins; Metal speciation; Inductively coupled plasma mass spectrometry; Marisa cornuarietis; and; Pomacea bridgesi; Sentinel organisms

Ultrasound wave-mediated enzyme-linked immunosorbent assay technique by Pragya Sharma; Pradip Nahar (pp. 241-246).

With the increasing burden of infectious diseases, it has become important to develop a rapid ELISA that could facilitate early diagnosis. Herein, we have shown that ultrasound waves can dramatically reduce the ELISA timing without losing its specificity or sensitivity. Ultrasound-mediated ELISA was best achieved on an activated microtiter plate which was able to covalently bind antigen or antibody in 10min when subjected to ultrasound waves in a sonicator bath having a temperature of 37°C, operating at a frequency of 40KHz and an output power of 120W. Blocking, antibody binding and secondary antibody–enzyme conjugate binding were also accomplished in 10min each in the sonicator bath under similar conditions. The validation of SELISA method was demonstrated by detecting IgE in allergic patient's sera. Total IgE detection by 40min-SELISA method gives similar absorbance value to that obtained by 20h conventional or 3h-HELISA procedure. As SELISA method can detect IgE even at the serum dilution of 1/50 (v/v) on photoactivated surface it could be significantly useful to confirm false negative cases. The inhibition assay ruled out the possibility of any cross reactivity or non-specific binding. As SELISA procedure is sensitive, specific and reproducible (intra- and inter-assay CVs were 9.65% and 8.47%) it could be an excellent alternative to conventional ELISA or HELISA procedures.

Keywords: Ultrasound waves; Sonication-mediated enzyme-linked immunosorbent assay; Photoactivation; 1-Fluoro-2-nitro-4-azidobenzene

Selective determination of dopamine with a cibacron blue/poly-1,5-diaminonaphthalene composite film by Adel A. Abdelwahab; Hak-Myoung Lee; Yoon-Bo Shim (pp. 247-253).

A selective detection method for dopamine (DA) was developed by incorporating cibacron blue (F3GA) into poly-1,5-diaminonaphthalene (PDAN) layer. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and cyclic voltammetry (CV) were employed to characterize the modified surfaces. The modified electrode was effective in selectively facilitating the electron transfer of DA and blocking the interferences of negatively charged species attributed to the sulfonate groups in the F3GA/PDAN composite film. This method enabled the determination of DA in the presence of various interfering species, including ascorbic acid (AA), in a phosphate buffer solution (pH 7.4). The modified electrode demonstrated good performance in the detection of DA in a concentration range of 5.0–100μM, with a detection limit ( k=3) of 0.1±0.01μM. The application was conducted for the determination of DA in a human urine sample and the sensor was proven to be rapid, has excellent selectivity, and stable amperometric response.

Keywords: Electrochemical sensor; Dopamine; Cibacron blue; Poly-1,5-diaminonaphthalene; Charge-selective film

A light-modulated chemosensor for methanol with ratiometry and colorimetry by Xu Zhang; Yi Chen (pp. 254-257).

A light-modulated chemosensor for methanol has been developed by using a photochromic spirophenanthoxazine as an indicator. In such a sensor, methanol can be detected easily by colorimetric recognition with high selectivity and anti-interference. Moreover, the concentration of indicator can be modulated and manipulated by light, which provides a simple way for the detection of methanol in a wide range of detectable limit that would be useful in different detection cases.

Keywords: Chemosensor; Methanol; Volatile organic compounds; Light-modulation; Ratiometry; Colorimetry

Detection of cationic guest molecules by quenching of luminescence of a self-assembled host molecule consisting of terbium(III) and calix[4]arene- p-tetrasulfonates by Takayuki Horiuchi; Nobuhiko Iki; Hitoshi Hoshino (pp. 258-263).

By self-assembly in aqueous solution, calix- (CAS) and thiacalix[4]arene- p-tetrasulfonate (TCAS) formed luminescent complexes Tb^III·(CAS)₂ and Tb^III·TCAS, respectively, which were utilized as a host for cationic guests. Addition of 1-ethylpyridinium guest quenched luminescence of Tb^III·(CAS)₂ in accordance with the Stern-Volmer (SV) relation with a low detection limit (D.L.) of 5.94×10⁻⁸M ( S/ N=3, M≡moldm⁻³). On the other hand, 1-ethylquinolinium quenched luminescence of Tb^III·TCAS most efficiently, affording a very low D.L. (6.71×10⁻¹⁰M). The agreement of the SV coefficients obtained with luminescent intensity ( K_SV,all=6.74×10⁶M⁻¹) and lifetime ( K_SV,Tb=6.50×10⁶M⁻¹) implied that dynamic quenching of⁵D₄ excited state of Tb^III was predominant in the quenching processes. The quenching rate was estimated to be k_q,Tb=9.94×10⁹M⁻¹s⁻¹, which was as fast as diffusion-limited rate. Quenching of Tb^III·(CAS)₂ was also applied to detection of NAD⁺, with a D.L. of 2.78×10⁻⁷M.

Keywords: Calixarenes; Host–guest systems; Self-assembly; Luminescence detection; Nicotineamide adenine dinucleotide

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Analytica Chimica Acta (v.650, #2)