Analytica Chimica Acta (v.596, #1)

Contents (v-vi).

Fast optical assessment of pesticide coverage on plants by Harald Hake; Ravid Ben-Zur; Israel Schechter; Angelika Anders (1-8).
This study addresses the necessity to provide fast assessment of pesticide coverage on plants, which is required for saving the expensive compounds and for protecting the environment against overdosing. The feasibility of applying several optical methods for direct detection of the pesticides on plants has been investigated. In order to overcome the matrix effect, which is related to plant fluorescence emission and to physiological spectral interferences, we suggest the application of a fluorescent labeling agent, in form of nano- and micro-crystallites. The coverage assessment is based on the spectral effects related to the interaction of these crystallites with the pesticides. Among the studied features are the fluorescence quenching/enhancement, spectral shifts and fluorescence lifetimes. It has been found that these spectral features are adequate for mapping the pesticides on plant surfaces, and assessing their coverage. Direct charge-coupled-device (CCD) fluorescence imaging was found as a low-cost adequate method, while Fourier transform spectral imaging provided more specific and accurate results.
Keywords: Pesticide; Detection; Plant; Laser; Fluorescence;

Fluorescence-based sensing of p-nitrophenol and p-nitrophenyl substituent organophosphates by Sheetal Paliwal; Melinda Wales; Theresa Good; Janet Grimsley; James Wild; Aleksandr Simonian (9-15).
A novel detection method for organophosphate neurotoxins has been described, based on the fluorescence quenching of a Coumarin derivative. These dyes are similar in structure to some organophosphates (OPs), and they fluoresce in the blue–green region of the spectra. This methodology has been utilized for the detection of organophosphates whose hydrolysis product is p-nitrophenol by using an enzyme, organophosphorus hydrolase (OPH). Coumarin1 in the presence of p-nitrophenol results in a quenching of fluorescence, providing a direct measure of the concentration of p-nitrophenol present in the sample. The decrease in fluorescence intensity is proportional to the paraoxon concentration in the range of 7.0 × 10−7–1.7 × 10−4  M. The specificity of this sensing application for p-nitrophenyl substituent OPs has also been demonstrated. OPs are a class of synthetic organic pesticides which generally have a short residual life and can cause numerous acute and chronic health effects. They have been an integral part of the agricultural industry for the past several decades due to their target specificities and selectable toxicities. The toxic nature of these compounds can be attributed to the species–specific inhibition of acetylcholinesterase (AChE), an important enzyme responsible for the regeneration of neural synaptic function. In addition to their wide agricultural and urban usage, they have also been exploited for the development of neurological chemical warfare agents. Currently available technologies for OP detection include sol–gel thin films, screen printed electrodes, acoustic patterning, gas chromatography–mass spectrometry, and various other intricate techniques that have limited field applicabilities. This optically-based approach promises much simpler and more direct detection capabilities.
Keywords: Organophosphates; Organophosphorus hydrolase; Coumarin1; Paraoxon; Parathion; Detection;

Study on the interaction between oxolinic acid aggregates and protein and its analytical application by Xia Wu; Jinhua Zheng; Honghong Ding; Dehuan Ran; Wei Xu; Yuanyuan Song; Jinghe Yang (16-22).
It was found that oxolinic acid (OA) at high concentration can self-assemble into nano- to micro- meter scale OA aggregates in Tris–HCl (pH 7.48) buffer solution. The nanoparticles of OA were adopted as fluorescence probes in the quantitative analysis of proteins. Under optimum conditions, the fluorescence quenching extent of nanometer scale OA aggregates was in proportion to the concentration of albumins in the range of 3.0 × 10−8 to 3.0 × 10−5  g mL−1 for bovine serum albumin (BSA) and 8.0 × 10−8 to 8.0 × 10−6  g mL−1 for human serum albumin (HSA). The detection limits (S/N = 3) were 3.4 × 10−9  g mL−1 for BSA, and 2.6 × 10−8  g mL−1 for HSA, respectively. Samples were satisfactorily determined. The interaction mechanism of the system was studied using fluorescence, UV–vis, resonance light scattering (RLS) and transmission electron microscope (TEM) technology, etc., indicating that the nonluminescent complex was formed between serum albumin molecular and OA, to disaggregate the self-association of OA, which resulted in the dominated static fluorescence quenching in the system.
Keywords: Fluorescence quenching; Oxolinic acid aggregates; Protein; Interaction study;

Simultaneous spectrophotometric flow-through measurements of pH, carbon dioxide fugacity, and total inorganic carbon in seawater by Zhaohui Aleck Wang; Xuewu Liu; Robert H. Byrne; Rik Wanninkhof; Renate E. Bernstein; Eric A. Kaltenbacher; James Patten (23-36).
An autonomous multi-parameter flow-through CO2 system has been developed to simultaneously measure surface seawater pH, carbon dioxide fugacity (fCO2), and total dissolved inorganic carbon (DIC). All three measurements are based on spectrophotometric determinations of solution pH at multiple wavelengths using sulfonephthalein indicators. The pH optical cell is machined from a PEEK polymer rod bearing a bore-hole with an optical pathlength of ∼15 cm. The fCO2 optical cell consists of Teflon AF 2400 (DuPont) capillary tubing sealed within the bore-hole of a PEEK rod. This Teflon AF tubing is filled with a standard indicator solution with a fixed total alkalinity, and forms a liquid core waveguide (LCW). The LCW functions as both a long pathlength (∼15 cm) optical cell and a membrane that equilibrates the internal standard solution with external seawater. fCO2 is then determined by measuring the pH of the internal solution. DIC is measured by determining the pH of standard internal solutions in equilibrium with seawater that has been acidified to convert all forms of DIC to CO2. The system runs repetitive measurement cycles with a sampling frequency of ∼7 samples (21 measurements) per hour. The system was used for underway measurements of sea surface pH, fCO2, and DIC during the CLIVAR/CO2 A16S cruise in the South Atlantic Ocean in 2005. The field precisions were evaluated to be 0.0008 units for pH, 0.9 μatm for fCO2, and 2.4 μmol kg−1 for DIC. These field precisions are close to those obtained in the laboratory. Direct comparison of our measurements and measurements obtained using established standard methods revealed that the system achieved field agreements of 0.0012 ± 0.0042 units for pH, 1.0 ± 2.5 μatm for fCO2, and 2.2 ± 6.0 μmol kg−1 for DIC. This system integrates spectrophotometric measurements of multiple CO2 parameters into a single package suitable for observations of both seawater and freshwater.
Keywords: Autonomous carbon measurements; Spectrophotometric; Carbon dioxide; pH; fugacity; Dissolved inorganic carbon;

An improved colorimetric method for chlorine dioxide and chlorite ion in drinking water using lissamine green B and horseradish peroxidase by Barry V. Pepich; Teri A. Dattilio; Patricia S. Fair; David J. Munch; Gilbert Gordon; Zsolt Körtvélyesi (37-45).
Lissamine Green B (LGB) was carefully selected as a potential candidate for the development of a new U.S. Environmental Protection Agency (EPA) method that is intended for use at water utilities to determine chlorine dioxide (ClO2) in drinking water. Chlorine dioxide reacts with LGB in aqueous solution to decrease the absorbance of LGB in direct proportion to the ClO2 concentration. LGB was confirmed to have adequate sensitivity, and to suffer less interference than other dyes reported in the literature. The stoichiometry for the reaction between LGB and ClO2 was found not to be 1:1 and is dependent on the LGB concentration. This required calibration of each LGB stock solution and prompted the investigation of alternate means of calibration, which utilized a horseradish peroxidase (HRP)-catalyzed conversion of chlorite ion (ClO2 ) to ClO2. This approach allowed the simultaneous determination of ClO2 concentration, which is also required each day at water plants that use ClO2. Studies were conducted to characterize and carefully optimize the HRP-conversion of ClO2 to ClO2 in order to yield reaction conditions that could be accomplished in less than 30 min at modest cost, yet meet EPA's sensitivity and robustness requirements for routine monitoring. An assessment of method detection limit, linearity and slope (or sensitivity), precision, and accuracy in finished drinking water matrices indicated that this approach was suitable for publication as EPA Method 327.0.
Keywords: Chlorine dioxide and chlorite analysis; Drinking water; Lissamine green B; Horseradish peroxidase;

In this work, the lightfastness of wool textile samples, dyed with madder and its principal components alizarin and purpurin, was investigated using two complementary experimental techniques: absorption and emission UV-vis spectroscopy and chromatography (HPLC-PDA). Spectroscopic techniques were used to follow the time course of ageing, whereas chromatography was applied to determine relative compositional changes that occurred after exposure of wool dyed samples to natural and artificial ageing. The results from the two techniques integrate well each other and provide complementary and useful indications about the sensitivity of the dyed textiles to ageing, showing that purpurin is the principal component responsible for the spectral and chromatic properties of madder as well as for its degradation. The fading of both the fibre and dye is reduced in the presence of alum and in the absence of oxygen. The multi-analytical approach used highlights the potential of the UV-vis spectroscopy for the investigation of dyes on textiles. The great sensitivity of the spectrofluorimetry makes this technique particularly promising for a non-destructive study of dyes on works of art.
Keywords: Madder (Rubia tinctorum); Alizarin; Purpurin; Ageing; UV-vis spectroscopy; Liquid chromatography (HPLC-PDA);

Interaction of brilliant red X-3B with bovine serum albumin and application to protein assay by Fang-Fang Chen; Shi-Long Wang; Xiang-Hu Liu; Ran Xu; Hong-Wen Gao (55-61).
The interaction of brilliant red X-3B (BRX) with bovine serum albumin (BSA) in three pH media has been characterized by the spectral correction technique. The binding number maximum of BRX was determined to be 102 at pH 2.03, 82 at pH 3.25 and 38 at pH 4.35 and the binding mechanism was analyzed in detail. The effects of ionic strength from 0 to 1 mol L−1 and temperature from 20 to 70 °C on the binding were investigated. The results showed that the interaction of BRX with BSA responded to the Langmuir adsorption isothermal model and the binding constant was determined. From the correlation between the binding number and the number of basic amino acid residues, the ion-pair attraction induced the union of non-covalent bonds including H-bond, van der Waals force and hydrophobic bond and the binding model was illustrated. The binding of BRX to BSA has resulted in change of the BSA conformation confirmed by means of circular dichroism. Using this interaction at pH 2.03, a sensitive method named the absorbance ratio difference spectrometry was established and applied to the protein assay and the limit of detection of protein was only 6 μg L−1. Two samples were determined and the results were in agreement with those obtained by the classical coomassie brilliant blue colorimetry.
Keywords: Brilliant red X-3B; Bovine serum albumin; Non-covalent binding; Conformational change; Molecular spectrometry; Protein assay;

A novel and sensitive cloud point extraction procedure for the determination of trace amounts of malachite green by spectrophotometry was developed. Malachite green was extracted at pH 2.5 mediated by micelles of nonionic surfactant Triton X-100. The extracted surfactant-rich phase was diluted with ethanol and its absorbance was measured at 630 nm. The effect of different variables such as pH, Triton X-100 concentration, cloud point temperature and time and diverse ions was investigated and optimum conditions were established. The calibration graph was linear in the range of 4–500 ng mL−1 of malachite green in the initial solution with r  = 0.9996 (n  = 10). Detection limit based on three times the standard deviation of the blank (3Sb) was 1.2 ng mL−1 and the relative standard deviation (R.S.D.) for 20 and 300 ng mL−1 of malachite green was 1.48 and 1.13% (n  = 8), respectively. The method was applied to the determination of malachite green in different fish farming and river water samples.
Keywords: Cloud point extraction; Malachite green; Spectrophotometry; Triton X-100;

This work describes the use of a multi-LED photometer for discrimination of mineral water samples, employing chromogenic reagents and chemometric techniques. Forty-five water samples (including 7 different brands of mineral water and samples of deionised, distilled and tap waters) were analysed in a monosegmented flow system, using three different chromogenic reagents (murexide, PAR and eriochrome black T) in a pH 10.0 NH3/NH4 + buffer in separate injections. Measurements were performed at 470, 500, 525, 562, 590, 612, 636 and 654 nm. Analyses were carried out using PCA, employing data sets including absorbance values obtained with one, two or all three reagents, which comprise 8, 16 or 24 variables, respectively. The best result was obtained with the data set from murexide and eriochrome black T, providing a clear distinction between 9 groups (distilled and deionised waters were classified in the same group). Based on the loading values, it was possible to select four wavelengths (470, 500, 590 and 654 nm) that provided a similar discrimination. With the use of these four LED, an HCA was performed, providing discrimination between 8 groups at a similarity level of 0.88. A model based on SIMCA allowed correctly classifying 94% of the samples. The discrimination between different groups is due to the metal ion contents in the water samples, mainly calcium and magnesium. Therefore, the use of common complexing reagents, such as murexide and erichrome black T, a multi-LED photometer and chemometric techniques provide an easy and simple method for water discrimination.
Keywords: Light emitting diode (LED); Photometry; Water; Pattern recognition; Principal component analysis; Hierarchical cluster analysis; Soft independent modelling of class analogy; Monosegmented flow;

In this paper, the electrogenerated chemiluminescence (ECL) from thiol-capped CdTe quantum dots (QDs) was reported. The ECL emission was occurred at −1.1 V and reached a maximum value at −2.4 V when the potential was cycled between 0.0 and −2.5 V. The reduced species of CdTe QDs could react with the coreactants to produce the ECL emission. The CdTe QD concentration (6.64 × 10−7  mol L−1) of ECL is lower than that (1.0 × 10−3  mol L−1) of chemiluminescence (CL). Based on the enhancement of light emission from thiol-capped CdTe QDs by H2O2 in the negative electrode potential, a novel method for the determination of H2O2 was developed. The light intensity was linearly proportional to the concentration of H2O2 between 2.0 × 10−7 and 1.0 × 10−5  mol L−1 with a detection limit of 6.0 × 10−8  mol L−1. Compared with most of previous reports, the proposed method has higher sensitivity for the determination of H2O2. In addition, the ECL spectrum of thiol-capped CdTe QDs exhibited a peak at around 620 nm, which was substantially red shifted from the photoluminescence (PL) spectrum, suggesting the surface states play an important role in this ECL process.
Keywords: Electrogenerated chemiluminescence; Thiol-capped CdTe quantum dots; Hydrogen peroxide;

A novel composite film modified glassy carbon electrode has been fabricated and characterized by scanning electron microscope (SEM) and voltammetry. The composite film comprises of single-wall carbon nanotube (SWNT), gold nanoparticle (GNP) and ionic liquid (i.e. 1-octyl-3-methylimidazolium hexafluorophosphate), thus has the characteristics of them. The resulting electrode shows good stability, high accumulation efficiency and strong promotion to electron transfer. On it, chloramphenicol can produce a sensitive cathodic peak at −0.66 V (versus SCE) in pH 7.0 phosphate buffer solutions. Parameters influencing the voltammetric response of chloramphenicol are optimized, which include the composition of the film and the operation conditions. Under the optimized conditions, the peak current is linear to chloramphenicol concentration in the range of 1.0 × 10−8–6.0 × 10−6  M, and the detection limit is estimated to be 5.0 × 10−9  M after an accumulation for 150 s on open circuit. The electrode is applied to the determination of chloramphenicol in milk samples, and the recoveries for the standards added are 97.0% and 100.3%. In addition, the electrochemical reaction of chloramphenicol and the effect of single-wall carbon nanotube, gold nanoparticle and ionic liquid are discussed.
Keywords: Chloramphenicol; Ionic liquid; Gold nanoparticles; Single-wall carbon nanotubes; Voltammetry;

Nanostructured progesterone immunosensor using a tyrosinase–colloidal gold–graphite–Teflon biosensor as amperometric transducer by Verónica Carralero; Araceli González-Cortés; Paloma Yáñez-Sedeño; José M. Pingarrón (86-91).
A novel progesterone immunosensor using a colloidal gold–graphite–Teflon–tyrosinase composite biosensor as amperometric transducer is reported. A sequential competitive configuration between the analyte and progesterone labelled with alkaline phosphatase (AP) was used. Phenyl phosphate was employed as the AP-substrate and the enzyme reaction product, phenol, was oxidized by tyrosinase to o-quinone, which is subsequently reduced at −0.1 V at the biocomposite electrode. Variables such as the concentration of phenyl phosphate, the amount of antibody attached to the electrode surface, immersion time in a 2% BSA solution, working pH and incubation times in progesterone and AP conjugate were optimized. A linear calibration graph for progesterone was obtained between 0 and 40 ng mL−1 with a slope value of −82.3 nA ng−1  mL, and a detection limit of 0.43 ng mL−1. The time needed to reach the steady-state current from the addition of phenyl phosphate was 30–40 s. These analytical characteristics improve substantially those reported for other progesterone immunosensors. A lifetime of 14 days with no need to apply any regeneration procedure was also achieved. The usefulness of the immunosensor was evaluated by determining progesterone in milk samples spiked with the analyte at 5.0 and 1.5 ng mL−1 concentration levels. Following a very simple procedure, involving only sample dilution, mean recoveries (n  = 7) of 98 ± 3% and 99 ± 3%, respectively, were obtained.
Keywords: Electrochemical immunosensor; Progesterone; Colloidal gold; Tyrosinase biosensor;

Poly(3,4-ethylenedioxythiophene-co-(5-amino-2-naphthalenesulfonic acid)) (PEDOT-PANS) film modified glassy carbon electrode was prepared by electrochemical polymerization technique. The properties of modified electrode was studied. It was found that the electrochemical properties of modified electrode was very much dependent on the experimental conditions, such as monomer oxidation potential and pH. The modified electrode surface was characterized by scanning electron microscopy (SEM). The PEDOT-PANS film modified electrode shows electrocatalytic activity toward oxidation of dopamine (DA) in acetate buffer solution (pH 5.0) and results in a marked enhancement of the current response. The linear sweep voltammetric (LSV) peak heights are linear with DA concentration from 2 × 10−6 to 1 × 10−5  M. The detection limit is 5 × 10−7  M. More over, the interferences of ascorbic acid (AA) and uric acid (UA) were effectively diminished. This work provides a simple and easy approach for selective determination of dopamine in the presence of ascorbic acid and uric acid.
Keywords: Poly(3,4-ethylenedioxythiophene-co-(5-amino-2-naphthalenesulfonic acid)); Linear sweep voltammetry; Dopamine; Ascorbic acid; Uric acid;

Positively charged gold nanoparticle (positively charged nano-Au), which was prepared, characterized by ξ-potential and transmission electron microscopy (TEM) was used in combination with l-cysteine to fabricate a modified electrode for electrocatalytic reaction of biomolecules. Compared with electrodes modified by negatively charged gold nanoparticle/l-cysteine, or l-cysteine alone, the electrode modified by the positively charged gold nanoparticle/l-cysteine exhibited excellent electrochemical behavior toward the oxidation of biomolecules such as ascorbic acid, dopamine and hydrogen peroxide. Moreover, the proposed mechanism for electrocatalytic response of positively charged gold nanoparticle was discussed. The immunosensor showed a specific to ascorbic acid in the range 5.1 × 10−7–6.7 × 10−4  M and a low detection limit of 1.5 × 10−7  M. The experimental results demonstrate that positively charged gold nanoparticle have more efficient electrocatalytic reaction than negatively charged gold nanoparticle, which opens up new approach for fabricating sensor.
Keywords: Positively charged gold nanoparticle; Electrocatalytic; Negatively charged gold nanoparticle;

Comparison of infrared-excited up-converting phosphors and europium nanoparticles as labels in a two-site immunoassay by Telle Ukonaho; Terhi Rantanen; Laura Jämsen; Katri Kuningas; Henna Päkkilä; Timo Lövgren; Tero Soukka (106-115).
Research in the field of immunoassays and labels used in the detection has been recently focused on particulate reporters, which possess very high specific activity that excludes the label as a sensitivity limiting factor. However, the large size and shape of the particulate labels may produce additional problems to immunoassay performance. The aim of this work was to study with two identical non-competitive two-site immunoassays whether up-converting phosphor (UCP) particles are comparable in performance with europium(III) chelate-dyed nanoparticles as particulate labels. In addition we strived to verify the common assumption of the photostability of up-converting phosphor particles supporting their potential applicability in imaging. Detection limits in two-site immunoassay for free prostate-specific antigen (free-PSA) were 0.53 ng L−1 and 1.3 ng L−1 using two different up-converting phosphors and 0.16 ng L−1 using europium(III) nanoparticle. Large size distribution and non-specific binding of up-converting phosphor particles caused assay variation in low analyte concentrations and limited the analytical detection limit. The non-specific binding was the major factor limiting the analytical sensitivity of the immunoassay. The results suggests the need for nanoscaled and uniformely sized UCP-particles to increace the sensitivity and applicability of up-converting phosphor particles. Anti-Stokes photoluminescence of up-converting phosphor particles did not photobleach when measured repeatedly, on the contrary, the time-resolved fluorescence of europium nanoparticles photobleached relatively rapidly.
Keywords: Particulate label; Up-converting phosphors (UCPs); Europium nanoparticles; Photobleaching;

Enzyme-linked immunosorbent assays for insulin-like growth factor-I using six-histidine tag fused proteins by Yong Huang; Ruina Shi; Xuefei Zhong; Dan Wang; Meiping Zhao; Yuanzong Li (116-123).
The fusion proteins of insulin-like growth factor-I (IGF-I) and six-histidine tag (IGF-I-6H, 6H-IGF-I-6H) were cloned, expressed, purified and renatured, with their immunoreaction properties and biological activities intact. The binding kinetics between these fusion proteins and anti-IGF-I antibody or anti-6H antibody were studied using surface plasmon resonance (SPR). Two enzyme-linked immunosorbent assay (ELISA) modes, which proved feasible in the measurement of human serum samples, were used to detect IGF-I with the help of the six-histidine tagged proteins. Furthermore, combining the production technique of the six-histidine tagged fusion protein with the competitive sandwich ELISA mode, using an enzyme labeled anti-6H antibody as a tracer, can be a universal immunochemical method to quantitate other polypeptides or proteins.
Keywords: Insulin-like growth factor-I; Six-histidine tag; Fusion protein; Enzyme-linked immunosorbent assay;

Investigation of single-site zirconium azaborolinyl complexes by laser desorption ionization time of flight mass spectrometry by Randall E. Duderstadt; Barbara M. Tsuie; Stephen F. Macha; Patrick A. Limbach (124-131).
Laser desorption ionization mass spectrometry (LDI-MS) was performed on selected azaborolinyl zirconium complexes in order to study them as potential industrial polyolefin catalyst compounds. UV–vis absorption data in conjunction with negative and positive ionization analyses revealed the mechanistic workings of the compounds when such single-site catalysts function in olefin polymerization reactions. Results presented highlight the many benefits of LDI-MS in the study of single-site zirconium azaborolinyl catalyst complexes including minimal sample preparation, the absence of matrix/pH/solvation effects, a lower degree of fragmentation as compared to EI ionization, high sensitivity and the ability to observe interesting and unique gas phase chemistry including ring slippage, and distinctive metal coordination and oxidation states. The extreme lability of the chlorine ligands was apparent, as well as the reactivity of the resulting zirconium metal center as demonstrated by the appearance of numerous similar four-coordinate zirconium species along with di-chloro-bridged dimers. LDI-MS permits analysis at low molecular weights, improving spectral characterization of these zirconium complexes.
Keywords: Laser desorption ionization mass spectrometry (LDI-MS); Zirconium; Azaboroline; Catalyst; Single-site;

Herein is reported, for the first time, a simple and reliable chiral reversed-phase liquid chromatographic method coupled to ultraviolet (UV) detection for simultaneous determination of eslicarbazepine acetate (ESL) and its metabolites, S-licarbazepine (S-LC), R-licarbazepine (R-LC) and oxcarbazepine (OXC), in mouse plasma and brain, liver and kidney tissue homogenates. All analytes and the internal standard were extracted from plasma and tissue homogenates by a solid-phase extraction procedure using Waters Oasis® hydrophilic–lipophilic balance cartridges. The chromatographic separation was performed by isocratic elution with water/methanol (88:12, v/v), pumped at a flow rate of 0.7 mL min−1, on a LichroCART 250-4 ChiraDex (β-cyclodextrin, 5 μm) column at 30 °C. The UV detector was set at 225 nm. Calibration curves were linear (r 2  ≥ 0.996) in the ranges 0.4–8 μg mL−1, 0.1–1.5 μg mL−1 and 0.1–2 μg mL−1 for ESL and OXC and in the ranges 0.4–80 μg mL−1, 0.1–15 μg mL−1 and 0.1–20 μg mL−1 for R-LC and S-LC in plasma, brain and liver/kidney homogenates, respectively. The overall precision not exceeded 11.6% (%CV) and the accuracy ranged from −3.79 to 3.84% (%bias), considering all analytes in all matrices. Hence, this method will be a useful tool to characterize the pharmacokinetic disposition of ESL in mice.
Keywords: Eslicarbazepine acetate; Oxcarbazepine; Mouse tissue samples; Enantioselective liquid chromatography; Bioanalytical method validation;

Determination of aflatoxins in olive oil by liquid chromatography–tandem mass spectrometry by Chiara Cavaliere; Patrizia Foglia; Chiara Guarino; Manuela Nazzari; Roberto Samperi; Aldo Laganà (141-148).
A liquid chromatography–tandem mass spectrometric with electrospray ionization (LC/ESI-MS/MS) method for determining the four naturally occurring aflatoxins (AFs) B1, B2, G1, and G2 in olive oil is proposed. AFs were extracted from oil sample by means of matrix solid phase dispersion (MSPDE), utilizing C18 as dispersing material. No further purification step, such as lipid removal, was performed. Aflatoxin M1, the hepatic metabolite of AFB1, was employed as internal standard. Olive oil extract was analyzed by LC/ESI-MS/MS in positive ionization mode, with multireaction monitoring acquisition. Due to a signal suppression ranging between 4 and 23%, quantitation was performed by matrix-matched calibration curves. The regression line coefficients of determination were above 0.9991. Sample recoveries ranged from 92 to 107%, with relative standard deviations below 13% for spiking levels between 0.5 and 5 ng g−1; method quantification limits ranged between 0.04 and 0.12 ng g−1. The developed LC/ESI-MS/MS method, although not as sensitive as LC coupled to fluorescence detection, is rapid, selective, accurate and precise, thus it can be used as confirmatory assay. The MSPDE appears suitable for application to other oleaginous matrices and for multiresidue investigation.
Keywords: Aflatoxins; Liquid chromatography–mass spectrometry; Matrix solid phase dispersion extraction; Mycotoxins; Olive oil; Tandem mass spectrometry;

Metabolic studies of mesterolone in horses by Emmie N.M. Ho; David K.K. Leung; Gary N.W. Leung; Terence S.M. Wan; Henry N.C. Wong; Xiaohua Xu; John H.K. Yeung (149-155).
Mesterolone (1α-methyl-5α-androstan-17β-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration. In vitro biotransformation studies of mesterolone were performed by incubating the steroid with horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid–liquid extraction and analysed by gas chromatography–mass spectrometry (GC–MS) after acylation or silylation. Five metabolites (M1–M5) were detected. They were 1α-methyl-5α-androstan-3α-ol-17-one (M1), 1α-methyl-5α-androstan-3β-ol-17-one (M2), 1α-methyl-5α-androstane-3α,17β-diol (M3), 1α-methyl-5α-androstane-3β,17β-diol (M4), and 1α-methyl-5α-androstane-3,17-dione (M5). Of these in vitro metabolites, M1, M3, M4 and M5 were confirmed using authentic reference standards. M2 was tentatively identified by mass spectral comparison to M1.For the in vivo metabolic studies, Proviron® (20 tablets × 25 mg of mesterolone) was administered orally to two thoroughbred geldings. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC–MS as described for the in vitro studies. The results revealed that mesterolone was extensively metabolised and the parent drug was not detected in urine. Three metabolites detected in the in vitro studies, namely M1, M2 and M4, were also detected in post-administration urine samples. In addition, two stereoisomers each of 1α-methyl-5α-androstane-3,17α-diol (M6 and M7) and 1α-methyl-5α-androstane-3,16-diol-17-one (M8 and M9), and an 18-hydroxylated metabolite 1α-methyl-5α-androstane-3,18-diol-17-one (M10) were also detected. The metabolic pathway for mesterolone is postulated. These studies have shown that metabolites M8, M9 and M10 could be used as potential screening targets for controlling the misuse of mesterolone in horses.
Keywords: Metabolism; Horse; Mesterolone; Steroids; Gas chromatography–mass spectrometry (GC–MS);

Ambient air monitoring with Auto-gas chromatography running in trigger mode by Liming Zhou; Yousheng Zeng; Pamela D. Hazlett; Valerie Matherne (156-163).
Speciated volatile organic compounds (VOC), either as ozone precursors or air toxics in the air, are commonly monitored by triggered canister method or continuous ozone precursor analyzer (commonly known as Auto-gas chromatography (GC)) method. In the triggered canister method, a canister sample is collected when a total non-methane organic compound (TNMOC) concentration exceeds a pre-determined trigger level. The canister sample is then analyzed in a lab in a later time. In the Auto-GC method, an online GC runs in a “continuous” mode with a sampling and analysis cycle of 1 h. Within the cycle hour, samples are collected only during the first 40 min.A new approach of Auto-GC running in trigger mode is developed in this study. This new approach uses Auto-GC but operates it in a trigger mode similar to the triggered canister sampling method. Compared to the triggered canister sample method, this system provides near real-time speciated VOC data, which are critical for responding to a high VOC concentration episode. Although the canister system generally costs less, its cost advantage may diminish if trigger events are frequent and the monitoring duration is long. Compared to continuous Auto-GC, triggered GC has its niche—it is better for capturing transient plumes with a small footprint. The continuous GC either misses a transient plume if the plume does not arrive at the sampling site during the sampling cycle or flattens the plume concentration peak by dilution with non-plume air sample.Field experience with this system for fenceline VOC monitoring is presented. The sampling and calibration strategy for trigger mode operation is described. The chromatograph retention time drift issues are discussed. The system performance is evaluated, including the method detection limit, precision and accuracy. The trigger mode configuration for VOC fenceline or near source monitoring in this work proved effective for local and transient plume identification.
Keywords: Ambient air monitoring; Ozone precursors; Air toxics; Speciated volatile organic compounds; Auto-gas chromatography; Canister sampling; Triggered Auto-gas chromatography;

This paper describes a new method for the determination of total flavonoids in Platycladus orientalis (L.) Franco. The method was based on dynamic microwave-assisted extraction (DMAE) coupled with on-line derivatization and ultraviolet–visible (UV–vis) detection. The influence of the experimental conditions was tested. Maximum extraction yield was achieved using 80% aqueous methanol of extraction solvent; 80 W of microwave output power; 5 min of extraction time; 1.0 mL min−1 of extraction solvent flow rate. The derivatization reaction between aluminium chloride and flavonoid is one of the most sensitive and selective reactions for total flavonoids determination. The optimized derivatization conditions are as follows: derivatization reagent 1.5% aluminium chloride methanol solution; reaction coil length 100 cm; derivatization reagent flow rate 1.5 mL min−1. The detection and quantification limits obtained are 0.28 and 0.92 mg g−1, respectively. The intra-day and inter-day precisions (R.S.D.) obtained are 1.5% and 4.6%, respectively. Mean recovery is 98.5%. This method was successfully applied to the determination of total flavonoids in P. orientalis (L.) Franco and compared with heat reflux extraction. The results showed that the higher extraction yield of total flavonoids was obtained by DMAE with shorter extraction time (5 min) and small quantity of extraction solvent (5 mL).
Keywords: Dynamic microwave-assisted extraction; On-line derivatization; Ultraviolet–visible detection; Total flavonoids; Platycladus orientalis (L.) Franco;

Exploring groundwater hydrochemistry of alluvial aquifers using multi-way modeling by Kunwar P. Singh; Amrita Malik; Sarita Sinha; Dinesh Mohan; Vinod Kumar Singh (171-182).
A three-way data set pertaining to hydrochemistry of the groundwater of north Indo-Gangetic alluvial plains was analyzed using three-way component analysis method with the purpose of extracting the information on spatial and temporal variation trends in groundwater composition. Three-way data modeling was performed using PARAFAC and Tucker3 models. The models were tested for their stability and goodness of optimal fit using core consistency diagnostic and split-half analysis. Although, a two-component PARAFAC model, explaining 50.47% of data variance, yielded 100% core consistency, it failed to qualify the validation test. Tucker3 model (3, 3, 1) captured 55.18% of the data variance and yielded simple diagonal core with three significant elements, explaining 100% of the core variability. Interpretation of the information obtained through Tucker3 model revealed that the groundwater quality in Khar watershed is mainly dominated by water hardness and related variables, whereas, water composition of the dug wells is dominated by alkalinity and carbonate/bicarbonates. Moreover, shallow groundwater sources in the region are contaminated with nitrate derived from fertilizers application in the region. The shallow aquifers are relatively more contaminated during the post-monsoon season.
Keywords: Multi-way modeling; Component analysis; PARAFAC; Tucker3; Groundwater; Hydrochemistry; Indo-Gangetic plains;