Analytica Chimica Acta (v.647, #2)

Application of computer-assisted molecular modeling for immunoassay of low molecular weight food contaminants: A review by Zhen-Lin Xu; Yu-Dong Shen; Ross C. Beier; Jin-Yi Yang; Hong-Tao Lei; Hong Wang; Yuan-Ming Sun (125-136).
Immunoassay for low molecular weight food contaminants, such as pesticides, veterinary drugs, and mycotoxins is now a well-established technique which meets the demand for a rapid, reliable, and cost-effective analytical method. However, due to limited understanding of the molecular structure of antibody binding sites and antigenic epitopes, as well as the intermolecular binding forces that come into play, the traditional ‘trial and error’ method used to develop antibodies still remains the method of choice. Therefore, development of enhanced immunochemical techniques for specific- and generic-assays, requires new approaches for antibody design that will improve affinity and specificity of the antibody in a more rapid and economic manner. Computer-assisted molecular modeling (CAMM) has been demonstrated to be a useful tool to help the immunochemist develop immunoassays. CAMM methods can be used to help direct improvements to important antibody features, and can provide insights into the effects of molecular structure on biological activity that are difficult or impossible to obtain in any other way. In this review, we briefly summarize applications of CAMM in immunoassay development, including assisting in hapten design, explaining cross-reactivity, modeling antibody–antigen interactions, and providing insights into the effects of the mouse body temperature on the three-dimensional conformation of a hapten during antibody production. The fundamentals and theory, programs and software, limitations, and prospects of CAMM in immunoassay development were also discussed.
Keywords: Food contaminants; Hapten design; Immunoassay; Molecular modeling;

Determination of phosphorus, sulfur and the halogens using high-temperature molecular absorption spectrometry in flames and furnaces—A review by Bernhard Welz; Fábio G. Lepri; Rennan G.O. Araujo; Sérgio L.C. Ferreira; Mao-Dong Huang; Michael Okruss; Helmut Becker-Ross (137-148).
The literature about the investigation of molecular spectra of phosphorus, sulfur and the halogens in flames and furnaces, and the use of these spectra for the determination of these non-metals has been reviewed. Most of the investigations were carried out using conventional atomic absorption spectrometers, and there were in essence two different approaches. In the first one, dual-channel spectrometers with a hydrogen or deuterium lamp were used, applying the two-line method for background correction; in the second one, a line source was used that emitted an atomic line, which overlapped with the molecular spectrum. The first approach had the advantage that any spectral interval could be accessed, but it was susceptible to spectral interference; the second one had the advantage that the conventional background correction systems could be used to minimize spectral interferences, but had the problem that an atomic line had to be found, which was overlapping sufficiently well with the maximum of the molecular absorption spectrum. More recently a variety of molecular absorption spectra were investigated using a low-resolution polychromator with a CCD array detector, but no attempt was made to use this approach for quantitative determination of non-metals. The recent introduction and commercial availability of high-resolution continuum source atomic absorption spectrometers is offering completely new possibilities for molecular absorption spectrometry and its use for the determination of non-metals. The use of a high-intensity continuum source together with a high-resolution spectrometer and a CCD array detector makes possible selecting the optimum wavelength for the determination and to exclude most spectral interferences.
Keywords: Molecular absorption spectrometry; Non-metals determination; Phosphorus; Sulfur; Halogens;

Multi-wavelength fingerprints of Cassia seed, a traditional Chinese medicine (TCM), were collected by high-performance liquid chromatography (HPLC) at two wavelengths with the use of diode array detection. The two data sets of chromatograms were combined by the data fusion-based method. This data set of fingerprints was compared separately with the two data sets collected at each of the two wavelengths. It was demonstrated with the use of principal component analysis (PCA), that multi-wavelength fingerprints provided a much improved representation of the differences in the samples. Thereafter, the multi-wavelength fingerprint data set was submitted for classification to a suite of chemometrics methods viz. fuzzy clustering (FC), SIMCA and the rank ordering MCDM PROMETHEE and GAIA. Each method highlighted different properties of the data matrix according to the fingerprints from different types of Cassia seeds. In general, the PROMETHEE and GAIA MCDM methods provided the most comprehensive information for matching and discrimination of the fingerprints, and appeared to be best suited for quality assurance purposes for these and similar types of sample.
Keywords: Multi-wavelength high-performance liquid chromatography fingerprinting; Cassia occidentalis L. seed; Classification; Chemometrics;

Fabrication of a novel capacitive immunosensor based on grafted ethylene diamine and self-assembled gold nanoparticle monolayer on glassy carbon electrode for the detection of Salmonella spp. is described for the first time. In the present study, the Salmonella spp. monoclonal antibodies (denoted as McAbs) was immobilized on gold nanoparticles. Interaction of McAbs and Salmonella spp. was detected directly using the electrochemical impedance spectroscopy (EIS) technique. The experimental results showed that the concentration of antigen was measured through the relative change in capacitance in the corresponding specific binding of Salmonella spp. and McAbs. Under the optimized conditions, the relative changes in capacitance were proportional to the logarithmic values of Salmonella spp. concentrations in the range of 1.0 × 102 to 1.0 × 105  CFU mL−1 (r  = 0.991) with the detection limit of 1.0 × 102  CFU mL−1. The stability of proposed immunosensor could be estimated by determining the relative change in capacitance, which remained almost the same in two months and decreased gradually to 85.3% of initial value after four months’ storage. The used immunosensor could be regenerated repeatedly by immersing in glycine–HCl buffer solution (pH 2.8). Finally, the proposed immunosensor was successfully used for the detection of Salmonella spp. in lab-processed commercial pork samples.
Keywords: Capacitive immunosensor; Gold nanoparticles; Electrochemical impedance spectroscopy; Salmonella spp.; Pork sample;

Evaluation of multi-walled carbon nanotubes as solid-phase extraction adsorbents of pesticides from agricultural, ornamental and forestal soils by M. Asensio-Ramos; J. Hernández-Borges; T.M. Borges-Miquel; M.A. Rodríguez-Delgado (167-176).
A new, simple and cost-effective method based on the use of multi-walled carbon nanotubes (MWCNTs) as solid-phase extraction stationary phases is proposed for the determination of a group of seven organophosphorus pesticides (i.e. ethoprophos, diazinon, chlorpyriphos-methyl, fenitrothion, malathion, chlorpyriphos and phosmet) and one thiadiazine (buprofezin) in different kinds of soil samples (forestal, ornamental and agricultural) using gas chromatography with nitrogen phosphorus detection. Soils were first ultrasound extracted with 10 mL 1:1 methanol/acetonitrile (v/v) and the evaporated extract redissolved in 20 mL water (pH 6.0) was passed through 100 mg of MWCNTs of 10–15 nm o.d., 2–6 nm i.d. and 0.1–10 μm length. Elution was carried out with 20 mL dichloromethane. The method was validated in terms of linearity, precision, recovery, accuracy and selectivity. Matrix-matched calibration was carried out for each type of soil since statistical differences between the calibration curves constructed in pure solvent and in the reconstituted soil extract were found for most of the pesticides under study. Recovery values of spiked samples ranged between 54 and 91% for the three types of soils (limits of detection (LODs) between 2.97 and 9.49 ng g−1), except for chlorpyrifos, chlorpyrifos-methyl and buprofezin which ranged between 12 and 54% (LODs between 3.14 and 72.4 ng g−1), which are the pesticides with the highest soil organic carbon sorption coefficient (K OC) values. Using a one-sample test (Student's t-test) with fortified samples at two concentration levels in each type of soil, no significant differences were observed between the real and the experimental values (accuracy percentages ranged between 87 and 117%). It is the first time that the adsorptive potential of MWCNTs for the extraction of organophosphorus pesticides from soils is investigated.
Keywords: Multi-walled carbon nanotubes; Solid-phase extraction; Pesticides; Carbon nanotubes; Soils; Gas chromatography; Nitrogen phosphorus detection;

Determination of organochlorine pesticides in water using dynamic hook-type liquid-phase microextraction by Pai-Shan Chen; Shih-Pin Huang; Ming-Ren Fuh; Shang-Da Huang (177-181).
We developed a simple and efficient headspace liquid-phase microextraction (LPME) technique named dynamic hook-type liquid-phase microextraction (DHT-LPME) and used it in combination with gas chromatography–mass spectrometry (GC–MS) and an electron capture detector (ECD). Aqueous specimens of organochlorine pesticides (OCPs) were used as model compounds to demonstrate the effectiveness of the technique. In the present study, the calibration curves were linear over at least 2 orders of magnitude with R 2 values of 0.997. The method detection limits (MDLs) varied from 2 to 44.0 ng L−1. The precision of DHT-LPME ranged from 6.5 to 14.4%. The relative recoveries of OCPs in rainwater were more than 84.2%. Enrichment factors (EF) in the range 275–1127 were obtained using DHT-LPME.
Keywords: Gas chromatography–mass spectrometry; Headspace; Dynamic hook-type liquid-phase microextraction; Organochlorine pesticides;

Ultrasound-assisted emulsification-microextraction (USAEME) procedure was developed for the determination of selected polychlorinated biphenyls (PCBs) in 10 mL of water samples by gas chromatography-mass-selective detection. After determination of the most suitable solvent and extraction time, several other parameters including solvent volume, centrifugation time and ionic strength of the sample were optimized using a 23 factorial experimental design. The optimized USAEME procedure used 200 μL of chloroform as extraction solvent, 10 min of extraction with no ionic strength adjustment at 25 °C and 5 min of centrifugation at 4000 rpm. The limits of detection ranged from 14 ng L−1 (for PCB153) to 30 ng L−1 (for PCB101). Recoveries of PCBs from fortified distilled water are over 80% for three different fortification levels between 0.1 and 5 μg L−1 and relative standard deviations of the recoveries are below 10%. The performance of the proposed method was compared with those involving traditional liquid–liquid extraction (LLE) and solid phase extraction (SPE) on the real water samples (i.e., tap and well water as well as domestic and industrial wastewaters, etc.) and comparable efficiencies were obtained. The proposed USAEME procedure has been demonstrated to be viable, simple, rapid and easy to use for residue analysis of PCBs in water samples.
Keywords: Polychlorinated biphenyls; Ultrasound-assisted emulsification-microextraction; Water analysis; Factorial design;

The oxidation of the recently synthesized Schiff base 3,6-bis((2-aminoethyl-5-Br-salicyliden)thio)pyridazine (PABST) with hydrogen peroxide was investigated using spectrophotometric studies. The reaction rate order and observed rate constant of the oxidation reaction was obtained in the mixture of N,N-dimethylformamide (DMF):water (30:70, v/v) at pH 10 using multivariate cure resolution alternative least squares (MCR-ALS) method and rank annihilation factor analysis (RAFA). The effective parameters on the oxidation rate constant such as percents of DMF, the effect of transition metals like Cu2+, Zn2+, Mn2+ and Hg2+ and the presence of surfactants were investigated. The keto-enol equilibria in DMF:water (30:70, v/v) solution at pH 7.6 was also investigated in the presence of surfactants. At concentrations above critical micelle concentration (cmc) of cationic surfactant cetyltrimethylammonium bromide (CTAB), the keto form was the predominant species, while at concentrations above cmc of anionic surfactant sodium dodecyl sulfate (SDS), the enol form was the predominant species. The kinetic reaction order and the rate constant of tautomerization in micellar medium were obtained using MCR-ALS and RAFA. The results obtained by both the methods were in a good agreement with each other. Also the effect of different volume percents of DMF on the rate constant of tautomerization was investigated. The neutral surfactant (Triton X-100) had no effect on tautomerization equilibrium.
Keywords: Catalytic effect; Keto-enol equilibria; Surfactants; Multivariate cure resolution alternative least squares; Rank annihilation factor analysis;

Fabrication of dissolved O2 metric uric acid biosensor using uricase epoxy resin biocomposite membrane by Jyoti Arora; Seep Nandwani; Manu Bhambi; C.S. Pundir (195-201).
Uricase purified from 20-day-old leaves of cowpea was immobilized on to epoxy resin membrane with 80% retention of initial activity of free enzyme and a conjugation yield of 0.056 mg/cm2. The uricase epoxy resin bioconjugate membrane was mounted over the sensing part of the combined electrode of ‘Aqualytic’ dissolved O2 (DO) meter to construct a uric acid biosensor. The biosensor measures the depletion of dissolved O2 during the oxidation of uric acid by immobilized uricase, which is directly proportional to uric acid concentration. The biosensor showed optimum response within 10–12 s at a pH 8.5 and 35 °C. A linear relationship was found between uric acid concentration from 0.025 to 0.1 mM and O2 (mg/l) consumed. The biosensor was employed for measurement of uric acid in serum. The mean value of uric acid in serum was 4.92 mg/dl in apparently healthy males and 3.11 mg/dl in apparently healthy females. The mean analytic recoveries of added uric acid in reaction mixture (8.9 and 9.8 mg/dl) were 93.6 ± 2.34 and 87.18 ± 3.17% respectively. The within and between batch CVs were <6.5 and <5.0%, respectively. The serum uric acid values obtained by present method and standard enzymic colorimetric method, showed a good correlation (r  = 0.996) and regression equation being y  = 0.984x  + 0.0674. Among the various metabolites tested only, glucose (11%), urea (38%), NaCl (25%) and cholesterol (13%) and ascorbic acid (56%) caused decrease, while, MgSO4 and CaCl2 had no effect on immobilized enzyme. The enzyme electrode showed only 32% decrease during its use for 100 times over a period of 60 days at 4 °C.
Keywords: Uric acid; Biosensor; Epoxy resin; Uricase; Dissolved O2 meter;

Single- and multi-analyte determination of gonadotropic hormones in urine by Surface Plasmon Resonance immunoassay by J. Treviño; A. Calle; J.M. Rodríguez-Frade; M. Mellado; L.M. Lechuga (202-209).
Single- and multi-analyte detection of two gonadotropic hormones (follicle stimulating hormone (hFSH) and luteinizing hormone (hLH)) was achieved by a Surface Plasmon Resonance (SPR) immunoassay on untreated human urine samples. Multi-analyte detection was accomplished using two alternative formats which are based in the individual or simultaneous immobilization of the hormones on the sensor surface. The lowest detection limit for both hormones in urine was found to be 1 ng mL−1, which in international units (IU) in terms of the World Health Organization (WHO) standards represents 8 mIU mL−1 of hLH and 14 mIU mL−1 of hFSH, respectively. The reliability of the assay was demonstrated by intra- and inter-assay variabilities <6%, chip-to-chip variabilities <5%, recoveries in the range of 80–120% and stability of the sensor response through more than 100 measurements. The sensitivity of this biosensing methodology renders it in a useful technique for the diagnosis of reproductive disorders, as well as for fertility monitoring.
Keywords: SPR; Immunosensor; Self-assembled monolayer; Human follicle stimulating hormone; Human luteinizing hormone; Point-of-care device; Urine samples;

A new ion-selective electrode (ISE) for the detection of trace chromium(III) was designed by using 2-acetylpyridine and nanoporous silica gel (APNSG)-functionalized carbon paste electrode (CPE). The presence of APNSG acted as not only a paste binder, but also a reactive material. With 7.5 wt% APNSG proportions, the developed electrode exhibited wide dynamic range of 1.0 × 10−8 to 1.0 × 10−3  M toward Cr(III) with a detection limit of 8.0 × 10−9  M and a Nernstian slope of 19.8 ± 0.2 mV decade−1. The as-prepared electrode displayed rapid response (∼55 s), long-time stability, and high sensitivity. Moreover, the potentiometric responses could be carried out with wide pH range of 1.5–5.0. In addition, the content of Cr(III) in food samples, e.g. coffee and tea leaves, has been assayed by the developed electrode, atomic absorption spectrophotometer (AAS) and atomic emission spectrometer (ICP-AES), respectively, and consistent results were obtained. Importantly, the response mechanism of the proposed electrode was investigated by using AC impedance and UV–vis spectroscopy.
Keywords: Carbon paste electrode; Potentiometric; Chromium; Silica gel;

High sensitive determination of zinc with novel water-soluble small molecular fluorescent sensor by Ying Weng; Zilin Chen; Fang Wang; Lin Xue; Hua Jiang (215-218).
A high sensitive method of quantitative analysis for the determination of zinc in the nutrition supplements has been developed by using a novel water-soluble fluorescent sensor HQ3: (8-pyridylmethyloxy-2-methyl-quinoline). Under the optimized condition of 67 mM phosphate buffer, pH 7.4, and 5% (v/v) DMSO, the zinc concentration showed good linear relationship with fluorescence intensity in the range of 7.5 × 10−8 to 2.5 × 10−5  M with the detection limit of 1.5 × 10−8  M. HQ3 exhibited high selectivity to zinc comparing with other metal ions except for cadmium. The developed analytical method was successfully used for determining the content of zinc in a real sample of zinc gluconate solution of Sanchine.
Keywords: Zinc ion; Fluorospectrophotometry; Quantitative analysis; 8-Hydroxyquinoline-based fluorescent sensor; Trace analysis;

A highly efficient capillary electrophoresis-based method for size determination of water-soluble CdSe/ZnS core–shell quantum dots by Yong-Qiang Li; Hai-Qiao Wang; Jian-Hao Wang; Li-Yun Guan; Bi-Feng Liu; Yuan-Di Zhao; Hong Chen (219-225).
This paper describes a highly efficient method for size determination of water-soluble CdSe/ZnS core–shell quantum dots (QDs) by capillary electrophoresis (CE) using polymer additive as sieving medium. The influence of some factors, such as kinds and concentrations of the sieving medium, pH, concentrations of the background electrolyte (BGE) and applied voltage, on the separation of QDs was investigated. Under the optimal separation conditions, four different sized QDs were successfully separated, and the relative standard deviation (RSD) of the migration times for these QDs was below 1.013%. In addition, an equation was fit by taking into account the correlation existing between the electrophoretic mobilities and the sizes of a set of QDs. The feasibility of this equation to measure the sizes of other QDs was confirmed by comparison with the sizes obtained by transmission electron microscopy (TEM) experiment. This work offers a novel method for size determination of QDs, and provides an important reference on the study of QDs based on CE.
Keywords: Capillary electrophoresis; Polymeric additive; Quantum dots; Sieving medium; Size determination; Size separation;

Development of high performance liquid chromatography method for buspirone in rabbit serum: Application to pharmacokinetic study by Ramesh Gannu; Shravan Kumar Yamsani; Chinna Reddy Palem; Vamshi Vishnu Yamsani; Harshini Kotagiri; Madhusudan Rao Yamsani (226-230).
A simple and sensitive high performance liquid chromatographic (HPLC) method for quantification of buspirone (BUSP) in rabbit serum was developed and validated. BUSP and internal standard (IS), diltiazem hydrochloride were extracted into dichloromethane and separated using an isocratic mobile phase, on a Kromasil C8 column. The eluent was monitored by UV detector at 235 nm and at a flow rate of 1.0 mL min−1. The linearity range of proposed method was 1–3000 ng mL−1. The intra-day and inter-day coefficient of variation and percent error values of the assay method were less than 15% and mean recovery was more than 97 and 96% for BUSP and IS, respectively. The method was found to be precise, accurate, and specific during the study. The method was successfully applied for pharmacokinetic study of buspirone after application of reservoir based transdermal therapeutic system of BUSP to rabbits.
Keywords: Buspirone; Reversed phase high performance liquid chromatography; Rabbit serum; Pharmacokinetics;

Investigation of the colourants used in icons of the Cretan School of iconography by Ioannis Karapanagiotis; Elpida Minopoulou; Lemonia Valianou; Sister Daniilia; Yannis Chryssoulakis (231-242).
The red shades of 13 icons (15th–17th century) of the Cretan School of iconography are investigated in detail to identify the inorganic and organic colouring materials comprising the paint layers. Examination of sample cross-sections is performed with optical microscopy. Micro-Raman spectroscopy and high performance liquid chromatography (HPLC) coupled to a photodiode array detector are employed for the identification of the inorganic and organic colouring materials, respectively. The results reveal the extensive use of coccid dyes by the Cretan painters: kermes (Kermes vermilio Planchon) is found in icons dated before the middle 16th century and cochineal in icons created several decades after the discovery of the New World. Other dyestuffs detected in the historical samples are madder (possibly Rubia tinctorum L., according to HPLC profiles), soluble redwood and indigoid dyes. Organic dyes were used by the painters as exclusive colouring matters (or glazes) or in mixtures with inorganic pigments, such as red ochre, cinnabar, minium, azurite lead white and carbon black.Liquid chromatography with mass spectrometry (LC–MS) coupled to a negative electrospray ionization mode is employed to provide information on the identity of some unknown colouring components, of the aforementioned dyes, detected in the historical samples. The results suggest that (i) the type B compound (also known as Bra′) is a dehydro-brazilein product and (ii) the deprotonated molecular ion of the type C compound corresponds to m/z  = 243. Both compounds are commonly used as markers for the identification of soluble redwood in historical samples. LC–MS analysis of cochineal shows that the dcIV and dcVII components are isomeric with carminic acid, as it has been recently suggested. Finally, LC–MS is employed to identify and record kermesic and flavokermesic acid in kermes and rubiadin in wild madder.
Keywords: Icon; Painting; Pigment; High performance liquid chromatography; Liquid chromatography-mass spectrometry; Raman spectroscopy;

Fungal secondary metabolites confer an indiscriminate resource of bioactive compounds with both pharmaceutical and industrial relevance. Synthesis of these compounds is tightly controlled through regulatory networks. These networks act in response to the environmental conditions in which the fungus grows. One class of fungal secondary metabolites that have drawn increased attention, is the polyketides produced by a high number of fungi and bacteria. The increasing number of publicly available genomic sequences of filamentous fungi, combined with new and highly efficient DNA-cloning techniques, has allowed the use of new efficient approaches to study the regulation and synthesis of these compounds. With the availability of new molecular genetic tools, a new bottleneck has occurred in fungal molecular biology; the analytical chemical analysis of the generated transgenic mutants. In this study, a method based on PARAFAC data analysis of HPLC data is presented and shown useful for gaining overview of complex and diverse datasets. Further, the strain specific and nutrient dependent regulation of polyketide synthesis is discussed.
Keywords: Fusarium; Secondary metabolites; Polyketide; Polyketide synthase; High Performance Liquid Chromatography; Parallel Factor Analysis; Global transcription factors;

Previously, we reported the DNA-inorganic hybrid material including double-stranded DNA by mixing the aqueous DNA solution and silane coupling reagents. Here, we immobilized the DNA-inorganic hybrid material onto the glass bead and prepared the DNA-immobilized glass bead column. The DNA-immobilized glass beads were stable in water and the amount of eluted DNA from the DNA-glass beads did not change for more than 1 week. Additionally, this DNA-immobilized column selectively accumulated the harmful compounds with the planar structure, such as dioxin- and polychlorinated biphenyl (PCB)-derivatives, and these accumulation percentages were 50–70%. Furthermore, the DNA-immobilized glass bead was recycled nine times by the application of ethanol solution and the accumulative ratio was maintained at more than 60% and did not appear to be decreasing. Therefore, these DNA-columns might have a potential for the selective removal and separation of DNA-intercalating molecules and harmful compounds with the planar structure from experimental or industrial drainages.
Keywords: DNA; Functional material; Intercalation; DNA-immobilized column; Organic–inorganic hybrid material;