Analytica Chimica Acta (v.658, #1)

The paper describes linear and nonlinear modeling of the wastewater data for the performance evaluation of an up-flow anaerobic sludge blanket (UASB) reactor based wastewater treatment plant (WWTP). Partial least squares regression (PLSR), multivariate polynomial regression (MPR) and artificial neural networks (ANNs) modeling methods were applied to predict the levels of biochemical oxygen demand (BOD) and chemical oxygen demand (COD) in the UASB reactor effluents using four input variables measured weekly in the influent wastewater during the peak (morning and evening) and non-peak (noon) hours over a period of 48 weeks. The performance of the models was assessed through the root mean squared error (RMSE), relative error of prediction in percentage (REP), the bias, the standard error of prediction (SEP), the coefficient of determination (R 2), the Nash–Sutcliffe coefficient of efficiency (E f), and the accuracy factor (A f), computed from the measured and model predicted values of the dependent variables (BOD, COD) in the WWTP effluents. Goodness of the model fit to the data was also evaluated through the relationship between the residuals and the model predicted values of BOD and COD. Although, the model predicted values of BOD and COD by all the three modeling approaches (PLSR, MPR, ANN) were in good agreement with their respective measured values in the WWTP effluents, the nonlinear models (MPR, ANNs) performed relatively better than the linear ones. These models can be used as a tool for the performance evaluation of the WWTPs.
Keywords: Wastewater; Partial least squares regression; Multivariate polynomial regression; Artificial neural networks; Modeling; Levenberg–Marquardt algorithm;

Antimony-film electrode for the determination of trace metals by sequential-injection analysis/anodic stripping voltammetry by Valéria Guzsvány; Hizuru Nakajima; Nobuaki Soh; Koji Nakano; Toshihiko Imato (12-17).
The possibility of applying antimony-film modified glassy carbon electrode in sequential-injection analysis (SIA) was investigated with the objective of determining Pb(II) and Cd(II) by anodic stripping voltammetry (ASV). The conditions of antimony-film deposition concerning composition of the plating/carrier solutions, concentrations of Sb(III) and hydrochloric acid, effects of different supporting electrolyte salts, and plating potential were optimized. It was found that the antimony-film deposition on glassy carbon substrate in a sample solution consisting of 750 μg L−1 Sb(III), 0.5 mol L−1 HCl at −1.5 V (vs. Ag/AgCl/3 mol L−1 KCl) yielded a modified electrode suitable for the determination of Pb(II) and Cd(II) at the μg L−1 level. The reproducibility of the analytical signals was characterized by a relative standard deviation lower than 2.8%, and the calculated values of detection limits were 1.2 μg L−1 for Pb(II) and 1.4 μg L−1 for Cd(II). The presence of KSCN in the sample solution offers the possibility of detecting ions with more negative oxidation potentials like Zn(II), Mn(II) or Cr(III). The developed SIA-ASV procedure was compared with the commonly used batch method, and its applicability was tested on a spiked tap water sample.
Keywords: Sequential-injection analysis; Anodic stripping voltammetry; Antimony-film electrode; Lead(II); Cadmium(II);

Application of thiophilic chromatography to deplete serum immunoglobulins in sample preparation for bidimensional electrophoresis by Francisco J. Salgado; Sara Vázquez; Alba Iglesias; Amparo Pérez-Díaz; Antonio Mera-Varela; Pilar Arias; Montserrat Nogueira (18-31).
Serum is a typical sample for non-invasive studies in clinical research. Its proteome characterization is challenging, since requires extensive protein depletion. Methods used nowadays for removal of high-abundance proteins are expensive or show quite often a low loading capacity, which has strong repercussions on the number of samples and replicates per analysis.In order to deplete immunoglobulins (Igs) and albumin (HSA) from 1 mL serum samples, we have developed a protocol based on a combination of thiophilic chromatography, not previously used in clinical proteomics, and a HSA-specific resin. Ig/HSA-depleted samples, immunoglobulinome and albuminone were analyzed by 2-DE. Thiophilic chromatography, coupled with HSA-depletion, allows a good 2-DE resolution as well as the visualization of new spots. Moreover, it yields enough protein to evaluate technical variability and facilitate subsequent protein identification. To validate the protocol, we carried out a preliminary comparative study between triplicate Igs/HSA-depleted serum samples from healthy control individuals and recently diagnosed/untreated rheumatoid arthritis (RA) patients. RA patients showed several acute phase proteins, as well as additional serum proteins, differentially and significantly regulated.Therefore, thiophilic chromatography can be used as an efficient and economical method in 2-DE to deplete immunoglobulins from large human serum samples before a more extensive fractioning.
Keywords: Albumin; Depletion; Immunoglobulin; Serum; Thiophilic chromatography; Bidimensional electrophoresis;

An analytical method is described for the determination of a broad range of emerging and priority pollutants, together with sewage molecular markers in environmental waters. The step-by-step study of the GC/MS analyses focuses on the effects of experimental variables using a large volume injection (LVI) technique [a programmed temperature-vaporising (PTV) inlet], the evaluation of a clean-up step using classical and newer sorbents (i.e. Al–N, Fl, NH2, PSA, Si, CN and DIOL), and the revision of how organic matter [i.e. humic acids (HA) content] affects method performance. Reproducibility and recoveries from spiked coastal water samples at different analyte concentrations (100, 250 and 500 ng L−1) as well as with different levels of spiked humic acids (2, 10 and 20 mg L−1) are reported indicating a good performance of the extraction procedure with low levels of HA (<10 mg L−1). The presence of HA is a critical parameter during the solid-phase extraction (SPE) procedures. Of the clean-up sorbents tested, CN and DIOL proved most efficient in cleaning-up the extracts with recoveries in the range of 66–77% and 100–114%, respectively for the selected analytes. Both GC/MS and PTV-GC/MS instrumental configurations were tested using final sewage effluents, riverine, estuarine and coastal water samples. However, limited applicability of the PTV inlet is reported for environmental applications, affording only a modest improvement in chromatographic signal-to-noise ratios.
Keywords: Pharmaceuticals and Personal Care Products; Endocrine disrupting chemicals; Molecular organic markers; Sewage; Transitional waters;

Dynamic flow-through extraction/fractionation methods have recently drawn much attention as appealing alternatives to the batchwise steady-state counterparts for the evaluation of environmentally available pools of potentially hazardous trace elements in solid matrices. The most critical weakness of flow-based column approaches lies in the small amount of solid that can be handled, whereby their applicability has been merely limited to date to the extraction of trace elements in highly homogeneous solid substrates; otherwise the representativeness of the test portion might not be assured.To tackle this limitation, we have devised an automated flow-through system incorporating a specially designed extraction column with a large volume capacity, wherein up to 2 g of solid sample could be handled without undue backpressure. The assembled flow setup was exploited for fast screening of potentially hazardous trace elements (namely, Cd, Cr, Cu, Pb, and Zn) in highly inhomogeneous municipal solid waste incineration (MSWI) bottom ashes. The pools of readily mobilizable metal forms were ascertained using the Toxicity Characteristic Leaching Procedure (TCLP) based on the usage of 0.1 mol L−1 CH3COOH as leachant and analysis of extracts by inductively coupled optical emission spectrometry. The application of a two-level full factorial (screening) design revealed that the effect of sample fluidization primarily but other experimental factors such as the solid to liquid ratio and extractant flow rate significantly influenced the leachability of given elements in raw bottom ashes at the 0.05 significance level.The analytical performance of the novel flow-based method capitalized on fluidized-bed extraction was evaluated in terms of accuracy, through the use of mass balance validation, reproducibility and operational time as compared to batchwise extraction and earlier flow injection/sequential injection microcolum-based leaching tests.
Keywords: Dynamic extraction; Fluidization; Trace element; Bottom ash; Bioaccessibility;

A modified aerospray apparatus was used to prepare a thin layer sample of matrix and analyte for quantitative analysis by MALDI-TOFMS. The apparatus consists of a set of coaxial tubing; the liquid sample is forced by a syringe pump through the inner capillary and it is nebulized by a flow of gas through the outer capillary. The small droplets of sample exiting the device are deposited onto a rotating plate, which serves as the sample surface for a time-of-flight mass spectrometer. An optimization was carried out after initial experiments with the device resulted in poorer than expected reproducibility of analyte signal. A two-level plus center point factorial experiment was performed investigating several factors, including the inner capillary internal diameter, gas pressure, liquid flow, spray distance, and time. After optimization the within-sample reproducibility of the analyte signal improved 3-fold, while the sample-to-sample reproducibility improved 4.5-fold.
Keywords: Optimization; Aerospray; Matrix-assisted laser desorption ionization; Sample preparation; Factorial design; Time-of-flight mass spectrometry;

A method in which antibodies are produced by using an immunogen heterogeneously conjugated with two or more kinds of haptens having unlike chemical structures against a same carrier protein was offered as an efficient approach for development of antibody to low molecular compounds. To appreciate the potentiality of the approach, 17β-estradiol (E2) and testosterone were selected as model compounds. The I 50 values of antiserum developed were 6 and 8 μg L−1 with the detection limits of 0.02 and 0.15 μg L−1 for E2 and testosterone, respectively. Antiserum owned an interesting characteristic that it was possible to independently analyze E2 and testosterone without mutual interference by making proper use of coating antigens. When using β-estradiol 17-hemisuccuinate (EH) conjugated with bovine serum albumin (BSA) as a coating antigen, the enzyme-linked immunosorbent assay (ELISA) was very selective to E2 and some estrogen analogues. Therefore, if testosterone coexisted in the ELISA for E2 detection, it showed no interference with it. From these findings, it was suggested that the verified method was an efficient and rational approach in development of polyclonal antibody to low molecular compounds.
Keywords: Low molecular compounds; Antibody production; Immunoassay; 17β-Estradiol (E2); Testosterone;

Optical waveguide sensor of volatile organic compounds based on PTA thin film by Renagul Abdurahman; Abliz Yimit; Hayrensa Ablat; Mamtimin Mahmut; Ji De Wang; Kiminori Itoh (63-67).
In this study, a sensitive optical waveguide (OWG) sensor for the detection and identification of volatile organic compounds (VOCs) was reported. The sensing membrane is constructed by immobilization of peroxopolytungsten acid (PTA) thin film over a single-mode potassium ion (K+) exchanged glass OWG by spin-coating method. A laser beam was coupled into and out of the glass optical waveguide using prism couplers, and dry air functioned as a carrier gas. The sensor was tested for various volatile organic compounds (VOCs), and it showed higher response to the chlorobenzene gas compared to other VOCs. Therefore, we used the OWG sensor to detect chlorobenzene gas as a typical example of VOCs. The sensor exhibits a linear response to chlorobenzene gas in the range of 0.4–1000 ppm with rapid response and good reversibility. The constructed sensor is easy to fabricate and it has some unique qualities which can be characterized as inexpensive, sensitive, and reusable.
Keywords: Gas sensor; Optical waveguide; Peroxopolytungsten acid; Chlolobenzene gas; VOCs;

A phosphate-selective electrode based on surfactant-modified zeolite (SMZ) particles into carbon-paste has been proposed (SMZ-CPE). The electrode was fully characterized in terms of composition, response time, ionic strength, thermal stability and usable pH range. The electrode containing 20% SMZ exhibited linear response range to phosphate species in the range of 1.58 × 10−5 to 1.00 × 10−2  M with a detection limit of 1.28 × 10−5  M and a Nernstian slope of 29.9 ± 0.9 mV per decade of phosphate concentration. The electrode response to phosphate remains constant in the pH range of 4–12 and in the presence of 1 × 10−4 to 4 × 10−3  M NaNO3. The response of the electrode reaches equilibrium within several seconds after immersing the electrode in phosphate solution. Common anions such as Cl, Br, I, NO3 , SO4 2− and Cr2O7 2− have little effect on the determination of phosphate but AsO4 3− shows some interference. A successful application of the electrode for determination of phosphate in a fertilizer, using direct potentiometry, is presented. The electrode was also used for the potentiometric titration of phosphate. The validation of the obtained results in each case was proved by statistical methods.
Keywords: Potentiometry; Phosphate determination; Surfactant-modified zeolite; Chemically modified carbon-paste electrode;

Hydrophilic biopolymer grafted on poly(dimethylsiloxane) surface for microchip electrophoresis by Jiu-Ju Feng; Ai-Jun Wang; Jing Fan; Jing-Juan Xu; Hong-Yuan Chen (75-80).
A novel covalent strategy was developed to modify the poly(dimethylsiloxane) (PDMS) surface. Briefly, dextran was selectively oxidized to aldehyde groups with sodium periodate and subsequently grafted onto amine-functionalized PDMS surface via Schiff base reaction. As expected, the coated PDMS surface efficiently prevented the biomolecules from adsorption. Electro-osmotic flow (EOF) was successfully suppressed compared with that on the native PDMS microchip. Moreover, the stability of EOF was greatly enhanced and the hydrophilicity of PDMS surface was also improved. To apply thus-coated microchip, the separation of peptides, protein and neurotransmitters was investigated in detail. For comparison, these analytes were also measured on the native PDMS microchips. The results demonstrated that these analytes were efficiently separated and detected on the coated PDMS microchips. Furthermore, the relative standard deviations of their migration times for run-to-run, day-to-day, and chip-to-chip reproducibilities were in the range of 0.6–2.7%. In addition, the coated PDMS microchips showed good stability within 1 month.
Keywords: Poly(dimethylsiloxane); Covalent modification; Microchip electrophoresis; Dextran;

Protein fingerprinting of Staphylococcus species by capillary electrophoresis with on-capillary derivatization and laser-induced fluorescence detection by Maria Teresa Veledo; Cristina Pelaez-Lorenzo; Ramon Gonzalez; Mercedes de Frutos; Jose Carlos Diez-Masa (81-86).
Capillary electrophoresis (CE) coupled with laser-induced fluorescence detection (LIF) has allowed to obtain protein fingerprints, which have demonstrated to be useful in microorganisms characterization. In this work, protein fingerprints of two species of Staphylococcus grown in different culture media and submitted to temperature and nitrosative stress were studied by CE-LIF. After the growth of the bacteria, protein extracts were obtained by cell lysis using sonication. The water-soluble fraction of these lysates was derivatized on-capillary with a fluorogenic dye, 3-(2-furoyl)quinoline-2-carboxaldehyde. The fluorescent products were analyzed by CE using phosphate buffer containing submicellar concentrations of sodium pentanesulfate and detected by LIF. Different protein fingerprints were obtained depending on the bacterial specie studied, indicating the usefulness of this method for the identification of different species of the same bacterial genus. It was also demonstrated that the CE protein fingerprints were dependent on the culture conditions, such as growth medium, or on stressing conditions, such as heat shock or nitrosative stress.
Keywords: Capillary electrophoresis; Laser-induced fluorescence detection; Protein fingerprinting; Staphylococcus; Growth conditions; Stressing conditions;

New approach for measurement of non-SHBG-bound testosterone in human plasma by Véronique Raverot; Jonathan Lopez; Catherine Grenot; Michel Pugeat; Henri Déchaud (87-90).
Testosterone (T) circulates in the blood tightly bound to sex hormone-binding globulin (SHBG) and weakly to albumin. Measuring protein unbound T (free) or non-SHBG-bound T rather than total T has been recommended for the evaluation of androgen disorders in humans. Ammonium sulfate precipitation has been widely used to separate [SHBG-T] complex from free and albumin-bound T. To achieve more specificity in this separation, we used monoclonal anti-SHBG antibody and developed a suitable and convenient immunoassay for measuring non-SHBG-bound T. Magnetic beads were covalently coupled to a monoclonal anti-SHBG antibody to capture [SHBG-T] complex from plasma samples. Magnetic separation was then performed to allow measurement of non-SHBG-bound T in the supernatant by direct radioimmunoassay. When 300 μL of plasma samples were incubated at room temperature with 10 μL of anti-SHBG beads, residual SHBG concentration was undetectable in the supernatant. The specificity of proteins retained on anti-SHBG beads was further demonstrated by peptide mass fingerprint on a MALDI-TOF analyzer. The non-specific adsorption of T on beads was low (5%), and dissociation of T from SHBG-T complex was less than 5% after 180 min of incubation. The plasma concentrations of non-SHBG-bound T using anti-SHBG beads were highly correlated to those obtained using ammonium sulfate precipitation. We conclude that SHBG immunocapture is a highly specific and useful tool for an experimental direct measurement of plasma non-SHBG-bound T. This methodology is also convenient and appropriate for routine and automated assay.
Keywords: Testosterone; Non-sex hormone-binding globulin-bound testosterone; Sex hormone-binding globulin; Ammonium sulfate; Hormone assay; Bioavailable testosterone;

Ribonucleotide and ribonucleoside determination by ambient pressure ion mobility spectrometry by Abu B. Kanu; Greg Hampikian; Simon D. Brandt; Herbert H. Hill (91-97).
Detection limits and reduced mobilities for 12 ribonucleotides and 4 ribonucleosides were measured by ambient pressure electrospray ionization–ion mobility spectrometry (ESI–IMS). With the instrument used in this study it was possible to separate some of these compounds within mixtures. Detection limits reported for ribonucleotides and ribonucleosides ranged from 15 to 300 pmol and the reduced mobilities ranged from 41 to 56 suggesting that ambient pressure ESI–IMS may be used for their rapid and sensitive separation and detection. This report demonstrates that it was possible to use ion mobility spectrometry (IMS) to obtain a spectrum for the separation of nucleotides and nucleosides in less than 1 min. The application holds great promise for nucleotide analysis in the area of separating DNA fragments in genome sequencing and also for forensics DNA typing examinations used for the identification of blood stains in crime scenes and paternity testing.
Keywords: Electrospray ionization; Ion mobility spectrometry; Nucleotides; Detection limit; Resolving power;

An improved reagent named 2-[2-(dibenzocarbazole)-ethoxy] ethyl chloroformate (DBCEC-Cl) for the determination of aliphatic amines by high-performance liquid chromatography (HPLC) with fluorescence detection and post-column online atmospheric chemical ionization-mass spectrometry (APCI-MS) identification has been developed. DBCEC-Cl could easily and quickly label aliphatic amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M+H]+ under APCI-MS in positive-ion mode. The ratios for fluorescence responses were I DBCEC-amine/I BCEC-amine  = 1.02–1.60; I DBCEC-amine/I BCEOC-amine  = 1.30–2.57; and I DBCEC-amine/I FMOC-amine  = 2.20–4.12 (here, I was relative fluorescence intensity). The ratios for MS responses were ICDBCEC-amine/ICBCEC-amine  = 4.16–29.31 and ICDBCEC-amine/ICBCEOC-amine  = 1.23–2.47 (Here, IC: APCI-MS ion current intensity). Detection limits calculated from 0.0244 pmol injection, at a signal-to-noise ratio of 3, were 0.3–3.0 fmol. The relative standard deviations for within-day determination (n  = 6) were 0.045–0.081% for retention time and 0.86–1.03% for peak area for the tested aliphatic amines. The mean intra- and inter-assay precision for all amine levels were <3.64% and 4.67%, respectively. The mean recoveries ranged from 96.9% to 104.7% with their standard deviations in the range of 1.80–2.70 (RSDs%). Excellent linear responses were observed with coefficients of >0.9991.
Keywords: Derivatization; Fluorescence; Amines; 2-[2-(Dibenzocarbazole)-ethoxy] ethyl chloroformate; Atmospheric chemical ionization-mass spectrometry;