Analytical Methods (v.5, #11)

Front cover (2637-2638).

Contents list (2639-2649).

New fluorescein-based H2O2 probe derivatization involves the simple dimethyl-thiocarbamoyl (DMTC) protecting group allowing for high aqueous ROS “turn-on” responses in the presence of Hg2+ concentration.

A coumarin-based chromogenic and ratiometric probe for hydrazine by Kun Li; Hao-Ran Xu; Kang-Kang Yu; Ji-Ting Hou; Xiao-Qi Yu (2653-2656).
A coumarin-based chromogenic and ratiometric probe for hydrazine was presented, which could selectively detect hydrazine via an intramolecular charge transfer (ICT) mechanism with a red-shift of 21 nm. Importantly, the probe could selectively detect hydrazine over other primary amines.

Separation and identification of phosphorothioate oligonucleotides by HILIC-ESIMS by Renee Easter; Colin Barry; Joseph Caruso; Patrick Limbach (2657-2659).
A combined hydrophilic interaction chromatography (HILIC) electrospray ionization mass spectrometry (ESI-MS) approach for the separation and identification of phosphorothioate oligonucleotides is described. Phosphorothioate 21-mer and 23-mer were separated by HILIC and detected using selected ion monitoring (SIM) ESI-MS. Phosphorothioates could be detected from 50 nM solutions suggesting effectiveness comparable to ion pairing reversed phase chromatography approaches.

The evaluation of forest fire severity and effect on soil organic matter based on the L*, a*, b* color reading system by Kazuto Sazawa; Takatoshi Wakimoto; Noriko Hata; Shigeru Taguchi; Shunitz Tanaka; Masamoto Tafu; Hideki Kuramitz (2660-2665).
The evaluation of forest fire severity and effect on soil is very important when discussing the damage to the environment. The purpose of this study is to evaluate the effect forest fires have on the soil color using the CIE Lab system. The color changes in four kinds of soil samples caused by heating at different temperatures were investigated. It was found that the Δa* and Δb* values drastically decrease when samples were heated over the temperature range from 200 to 250 °C, that is above the ignition temperature of volatile matter. The Δa* and Δb* values show a good linear negative correlation with the atomic ratios of H/C and O/C. The results obtained from this study indicate that the proposed method is able to evaluate the dehydration and decarboxylation of soil caused by forest fires. We demonstrated this method using soil samples collected from unburned and burned areas in Kalimantan, Indonesia.

Direct determination of transition metals in mussel tissue digests using high-performance chelation ion chromatography with monolithic silica based chelating ion exchangers by Nicola McGillicuddy; Ekaterina P. Nesterenko; Phil Jones; Dario Caldarola; Barbara Onida; Ashley T. Townsend; Dimitar P. Mitev; Pavel N. Nesterenko; Brett Paull (2666-2673).
A new high performance chelation ion chromatography method was developed for the direct separation and detection of selected transition metals in shellfish (mussel) tissue digest samples. Using two different bonded silica monolithic chelating stationary phases, exhibiting differing selectivities for metals of interest, were applied within the method. The bonded chelating chemistries were N-hydroxyethyliminodiacetic (HEIDA) and N-(2-hydroxyethyl)-N-(2-[phosphonomethyl)amino]acetic (HEPMA) acids. Quantitative determination of Mn(ii), Cd(ii) and Zn(ii) concentrations within shellfish tissue (Mytilus edulis), following microwave assisted acid digestion. Post-column detection was achieved using spectophotometric detection at 510 nm after reaction with 4-(2-pyridylazo) resorcinol. The eluents employed were 0.5 M KNO3, pH 2.4, for the HEIDA-bonded stationary phase, and 0.1 M KNO3, pH 2.6, for the HEPMA-bonded monolithic column. The HEPMA phase provided improved resolution between Mn(ii) and interfering alkaline earth metal cations compared to the HEIDA-bonded phase. Concentrations of metals were determined using standard addition, and Cd(ii), Mn(ii), and Zn(ii) cations were detected at <10 μg g−1, <50 μg g−1 and <700 μg g−1, respectively. Sample analysis using sector field inductively coupled plasma-mass spectrometry (ICP-MS) was carried out to generate comparative data to that obtained using the chromatographic method.

The dissolution determination of multiple components in herbal preparations is a crucial method for controlling the quality of oral solid dosage forms. In order to improve the low detection sensitivity of dissolution samples, an efficient method for the extraction and concentration of multiple components in dissolution samples of Huoxiang Zhengqi tablets was developed by partitioned dispersive liquid-liquid microextraction (PDLLME). In the PDLLME process, 1.6 mL of THF as the disperser solvent and 100 μL of 1,2-dichloroethane as the extraction solvent were injected into 8 mL of the dissolution sample solution. Based on the partition coefficient, a portion of THF will transfer into the extraction solvent, then the multiple components in the Huoxiang Zhengqi tablet can be extracted into the mixture extraction phase of 1,2-dichloroethane and partitioned THF. In combination with HPLC-UV analysis, the enrichment factors reached to 43 to 119-fold for five of the target components (three of the more polar components were unknown). For honokiol and magnolol, a linear range from 10 to 1000 μg L−1 was obtained, and the recoveries at three spiking levels ranged from 90.2 to 99.4% with RSD less than 7.2% (n = 3). The proposed method may be applied for the extraction and concentration of multiple components in the dissolution samples of other herbal preparations.

Identification of bacteria in drinking water with Raman spectroscopy by Jack van de Vossenberg; Heli Tervahauta; Kees Maquelin; Carola H. W. Blokker-Koopmans; Marijan Uytewaal-Aarts; Dick van der Kooij; Annemarie P. van Wezel; Bram van der Gaag (2679-2687).
Raman spectroscopy was used to discriminate between Legionella strains and between E. coli and coliform strains. The relationship between triplicate Raman spectra derived from Legionella bacteria was compared with that derived from a blind set of samples and amplified fragment length polymorphism (AFLP) data from the same strains. Triplicate Raman spectra of E. coli and coliform bacteria were compared with their 16S phylogeny. In all cases Raman spectra were reproducible and could be distinguished from spectra of other organisms down to the strain level. All samples in a blind fourth set were identified correctly. Raman spectra of organisms of the same coliform species clustered according to 16S rRNA gene phylogeny, except for Enterobacter spp. At higher taxonomic levels the relationship between species was less comparable. For Legionella strains the Raman spectra grouped according to AFLP groups, based on the dataset used in this study. Raman spectroscopy could correctly distinguish E. coli from other coliform bacteria and L. pneumophila from non-pneumophila strains. Incubation of Legionella strains in different types of drinking water at different temperatures over a period of one week introduced so little variation in the Raman spectra that only very closely related L. pneumophila strains could not be distinguished from each other. Temperature, ageing and water type did not influence the identification potency of Raman spectroscopy in all cases. Given the accuracy, speed and simplicity of the Raman spectroscopy technique this method seems a welcome addition to the current tools for identification of waterborne bacteria.

A resonance Rayleigh scattering detection of DNA hybridization based on interaction between DNA and surfactants by Ying Ma; Changqun Cai; Lin Luo; Jiaqi Xie; Xiaoming Chen (2688-2693).
We developed a new method that used surfactants to detect DNA hybridization in aqueous media, this method is highly sensitive and based on a resonance Rayleigh scattering (RRS) technique. Cationic surfactants, cetyltrimethyl ammonium bromide (CTAB) and cetylpyridinebromide (CPB), anionic surfactants, sodium dodecylbenzene sulphonate (SDBS) and sodium dodecyl sulphonate (SDS), nonionic surfactants Triton-100 (TX-100) and Tween-80 (T-80), interacted with double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), and were investigated respectively. The RRS signal was strongest in the case of the CTAB complex which therefore was selected as the probe. Based on the RRS data, a linear relation between RRS intensity and target DNA concentration was found. Mechanism investigations have shown that CTAB can bind to DNA by electrostatic and hydrophobic interactions and aggregate on the molecular surface of dsDNA at pH 7.2. By using CTAB as a probe, we detected and analyzed two kinds of characteristic sequences.

Protein–polyphenol interaction on silica beads for astringency tests based on eye, photography or reflectance detection modes by J. Rafaela L. Guerreiro; Duncan S. Sutherland; Victor De Freitas; M. Goreti F. Sales (2694-2703).
Astringency is an organoleptic property of beverages and food products resulting mainly from the interaction of salivary proteins with dietary polyphenols. It is of great importance to consumers, but the only effective way of measuring it involves trained sensorial panellists, providing subjective and expensive responses. Concurrent chemical evaluations try to screen food astringency, by means of polyphenol and protein precipitation procedures, but these are far from the real human astringency sensation where not all polyphenol–protein interactions lead to the occurrence of precipitate. Here, a novel chemical approach that tries to mimic protein–polyphenol interactions in the mouth is presented to evaluate astringency. A protein, acting as a salivary protein, is attached to a solid support to which the polyphenol binds (just as happens when drinking wine), with subsequent colour alteration that is fully independent from the occurrence of precipitate. Employing this simple concept, Bovine Serum Albumin (BSA) was selected as the model salivary protein and used to cover the surface of silica beads. Tannic Acid (TA), employed as the model polyphenol, was allowed to interact with the BSA on the silica support and its adsorption to the protein was detected by reaction with Fe(iii) and subsequent colour development. Quantitative data of TA in the samples were extracted by colorimetric or reflectance studies over the solid materials. The analysis was done by taking a regular picture with a digital camera, opening the image file in common software and extracting the colour coordinates from HSL (Hue, Saturation, Lightness) and RGB (Red, Green, Blue) colour model systems; linear ranges were observed from 10.6 to 106.0 μmol L−1. The latter was based on the Kubelka–Munk response, showing a linear gain with concentrations from 0.3 to 10.5 μmol L−1. In either of these two approaches, semi-quantitative estimation of TA was enabled by direct eye comparison. The correlation between the levels of adsorbed TA and the astringency of beverages was tested by using the assay to check the astringency of wines and comparing these to the response of sensorial panellists. Results of the two methods correlated well. The proposed sensor has significant potential as a robust tool for the quantitative/semi-quantitative evaluation of astringency in wine.

Methods to determine neurochemical concentrations in small samples of tissue are needed to map interactions among neurotransmitters. In particular, correlating physiological measurements of neurotransmitter release and the tissue content in a small region would be valuable. HPLC is the standard method for tissue content analysis but it requires microliter samples and the detector often varies by the class of compound being quantified; thus detecting molecules from different classes can be difficult. In this paper, we develop capillary electrophoresis with fast-scan cyclic voltammetry detection (CE-FSCV) for analysis of dopamine, serotonin, and adenosine content in tissue punches from rat brain slices. Using field-amplified sample stacking, the limit of detection was 5 nM for dopamine, 10 nM for serotonin, and 50 nM for adenosine. Neurotransmitters could be measured from a tissue punch as small as 7 μg (7 nL) of tissue, three orders of magnitude smaller than a typical HPLC sample. Tissue content analysis of punches in successive slices through the striatum revealed higher dopamine but lower adenosine content in the anterior striatum. Stimulated dopamine release was measured in a brain slice, then a tissue punch collected from the recording region. Dopamine content and release had a correlation coefficient of 0.71, which indicates much of the variance in stimulated release is due to variance in tissue content. CE-FSCV should facilitate measurements of tissue content in nanoliter samples, leading to a better understanding of how diseases or drugs affect dopamine, serotonin, and adenosine content.

The detailed electrochemical character of brucine at a poly(aspartic acid)-modified electrode and its sensitive determination by Jie Liu; Lingxi Yang; Kai Zhang; Kunjing Li; Xianli Wu; Baoxian Ye (2712-2719).
A simple and reliable electrochemical sensor based on poly(dl-aspartic acid)-modified glassy carbon electrode was proposed for direct determination of brucine. The electrochemical character of brucine on this sensor was carefully and systematically studied by cyclic voltammetry. Some kinetic parameters were calculated and a reasonable reaction mechanism for brucine at the poly(dl-aspartic acid)/GCE was also proposed. Meanwhile, a new electroanalytical method for determination of brucine was proposed with a highly sensitive detection limit of 3 × 10−8 mol L−1 (S/N = 3) and a wide linear range of 5.0 × 10−8 to 9.0 × 10−5 mol L−1. More importantly, this sensor exhibited good stability and excellent reproducibility in the response for brucine.

A competitive immunoassay based on gold nanoparticles for the detection of chromium in water samples by Junhui Zou; Yong Tang; Yifan Zhai; Hui Zhong; Jianxun Song (2720-2726).
A competitive enzyme-linked immunosorbent assay (ELISA) based on colloidal gold nanoparticles modified with a monoclonal antibody and horseradish peroxidase (HRP) was developed and validated for the quantitative determination of chromium in water samples. The monoclonal antibody against Cr(iii)–EDTA secreted by the hybridoma cell line 5G12C5 was prepared through cell fusion. The ELISA conditions were optimized using the chessboard method and the sensitivity and specificity of this assay were determined. The results show that this competitive immunoassay has a half maximum inhibition concentration (IC50) of 3.85 ng mL−1 and a limit of detection (LOD) of 0.1 ng mL−1. Specificity assays revealed that the cross-reactivities (CRs) of this assay with 10 other common metal ions were all below 1%. The accuracy of this immunoassay was confirmed by using certified reference materials (GSBZ50027-94, GSB07-1187-2000). Furthermore, the assay was used to detect the concentrations of Cr(vi) and total chromium in local surface water samples after the pretreatment procedure. The data show that the results of this method correlated well with those obtained from atomic absorption spectroscopy (AAS) (R2 = 0.9728), and that this method is capable of detecting the amount of total chromium and Cr(vi).

Metabolomics, the global analysis of small molecules in a biological system in a holistic context, has played increasingly important roles in studies on the potential mechanisms of drugs. Modified Sinisan (MSNS), a traditional Chinese medicine, has showed good clinical efficacy in the treatment of liver injury, and its mechanism remains unclear. The present study was undertaken to explore the metabolomic characters of liver injury induced by dimethylnitrosamine and the therapeutic effects of MSNS. The study utilized ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC/ESI-Q-TOF-MS) in positive electrospray ionization combined with pattern recognition approach including principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA) to demonstrate comprehensive metabolic characteristics and discover differentiating metabolites. Significant changes of 11 biomarkers in rat urine were identified and they were associated with perturbations in riboflavin metabolism, arginine and proline metabolism, histidine metabolism, sphingolipid metabolism, alanine, aspartate and glutamate metabolism, cysteine and methionine metabolism and pyrimidine metabolism. Of note, MSNS administration could provide satisfactory effects on liver injury through partially reversing the level of biomarkers in the urine of liver injured rats. These results show the power of metabolomics in unraveling the potential mechanisms of MSNS and may help us to obtain a better understand of the underlying pathophysiological processes of liver injury.

A very simple and sensitive method was developed for the simultaneous determination of carbamate and organophosphorus pesticides in water samples, using dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography-mass spectrometry (GC-MS). Carbofuran and chlorfenvinphos were used as model molecules in this study. The optimisation of some important DLLME parameters (type and volume of extraction and dispersive solvents, sample volume, extraction time, and use of NaCl) was performed using a design of experiments approach. Under optimised conditions of the DLLME methodology (500 μL of methanol, 80 μL of chlorobenzene, 10 mL of aqueous sample, 4 minutes of vortexing and no salt addition), the calibration curve was linear in the concentration range of 0.1–250 μg L−1 for carbofuran and 0.1–200 μg L−1 for chlorfenvinphos. The limits of detection (LODs) were 0.04 μg L−1 for the carbamate and 0.02 μg L−1 for the organophosphorus pesticide. The proposed method was successfully applied to the analysis in river, mineral and tap water samples, yielding satisfactory results.

The international standard method for the determination of volatile organic compounds (VOCs) emitted from building materials and other products used in indoor environments, ISO 16000-6:2011, specifies use of the sorbent Tenax TA. This sorbent does not allow quantifiable determination of very volatile organic compounds (VVOCs), but an informative annex to the latest version of this standard, recommends the use of multi-sorbent tubes in order to extend the volatility range of the method to include more volatile compounds. There is a need to investigate the extent of the improvement possible using these multi-sorbent tubes and also to check that the performance for compounds which are normally detected on the single sorbent tube is not compromised. Screening tests of material emissions and Nalophan bags containing atmospheres of a mixture of VVOCs of interest have been applied to compare the performance of tubes containing Tenax TA with those containing a multi-sorbent tube (quartz wool/Tenax TA/Carbograph 5TD). A significant improvement was found using the multi-sorbent tube, with no breakthrough for six VVOCs up to a sample volume of at least 10 litres, compared with maximum safe sample volumes for these compounds using Tenax TA alone ranging from <200 ml to 3.5 litres. More modest improvements were identified for a further 11 compounds.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is known as a highly toxic environmental contaminant and poses potential risks to human and animal health. As a persistent organic pollutant, 2,3,7,8-TCDD is hard to metabolize and thus gets accumulated in the human and animal body. Literature reports on the metabolism study of 2,3,7,8-TCDD are rare. The traditional method of GC-MS for 2,3,7,8-TCDD analysis might not be amenable for the direct analysis of its polar metabolites. In this study, in vitro metabolism of 2,3,7,8-TCDD with rat liver microsomes by using LC-MS and MS/MS was investigated and two hydroxylated metabolites of 2,3,7,8-TCDD and four trichloro-dihydroxydibenzo-p-dioxins were identified from the direct LC-MS and MS/MS analyses of the incubated samples.

Various independent component analysis (ICA) algorithms (MILCA, JADE, SIMPLISMA, RADICAL) are applied for simultaneous spectroscopic determination of two groups of transition metals: Co(ii)–Fe(iii)–Cu(ii)–Zn(ii)–Ni(ii) and Pt(iv)–Pd(ii)–Ir(iv)–Rh(iii)–Ru(iii)) in complex mixtures. The analysis is based on the decomposition of spectra of multicomponent mixtures in the UV-VIS region based on the natural absorbance of metal salts, or, when a better sensitivity is desirable, based on the absorbance of their complexes with 4-(2-pyridylazo)resorcinol (PAR) and ethylenediaminetetraacetic acid (EDTA). Good quality spectral resolution of up to seven-component mixtures was achieved (correlation coefficients between resolved and experimental spectra are not less than 0.90). In general, the relative errors in the recovered concentrations are at levels of only several percent. While being superior to other ICA algorithms, MILCA is comparable or even outperforms other classical chemometric methods for quantitative analysis that were used for comparison purposes (Partial Least Squares (PLS), Principal Component Regression (PCR), Alternating Least Squares (ALS)). Simultaneous quantitative analysis is possible for mixtures containing up to five metals in the broad concentration ranges even when individual spectra show 99% overlap. A small excess of derivatization reagent (till threefold excess to the sum of metal concentrations) is optimal to obtain good quantitative results. The proposed method was used for analysis of authentic samples (multimineral supplements and platinum concentrates). The resolved ICA concentrations match well with the labelled amounts and the results of other chemometric methods (ALS, PLS). ICA decomposition considerably improves the application range of spectroscopy for metal quantification in mixtures.

Conformational dynamics of DNA bulge loops investigated by CE-LIF by Ming-Feng Huang; Huan-Tsung Chang (2773-2777).
Capillary electrophoresis with laser induced fluorescence (CE-LIF) detection has been utilized to investigate the metastable states of DNA bulge loops during strand displacement reactions. DNA samples consisting of one of the oligonucleotides with sequences of X(CAG)nY (n = 4–8, X = CACGAGCACGC and Y = CACACCGAAGC) and a 22-mer oligonucleotide with a sequence of Y′X′ (Y′ = GCGTGCTCGTG and X′ = GCTTCGGTGTG) were separated within 7 min by CE-LIF using 2.7% poly(ethylene oxide) (PEO) in the presence of EOF. Our CE data allowed calculation of the equilibrium constant (K) of the ds-DNA of 34-mer X(CAG)4Y and 22-mer Y′X′ at 25 °C to be 1.3 (±0.1) × 109 M−1, with a rate constant (k) of 3.7 (±0.4) × 105 M−1 s−1. We have further applied this CE-LIF technique to separately study the strand displacement of a ds-DNA of the 34-mer X(CAG)4Y and 22-mer Y′X′ bulge loop DNA with a 22-mer XY and that of a ds-DNA of 22-mer XY and Y′X′ with the 34-mer X(CAG)4Y. Our study reveals that the metastable DNA bulge loops and unbinding oligonucleotides were coexistent at room temperature, while they underwent displacement during a heating/cooling cycle. Our study reveals that this CE-LIF approach has great potential for the investigation of DNA related biological functions.

In this work, a novel molecularly imprinted polymer (MIP) coated stir bar was fabricated and used for the extraction of trace amounts of Cu from drinking water. Cu–morin complex was used as template molecule which was chemically bonded to a glass stir bar and was employed for stir bar sorptive extraction of trace amounts of copper ions. The effects of different parameters such as pH, adsorption and desorption time, stirring rate, temperature and amount of ligand (morin) were evaluated on the extraction efficiency and the optimum conditions were established: the extraction and desorption times were fixed respectively at 20 and 30 min, mole ratio of ligand to the analyte was selected as 2, stirring speed was 700 rpm, pH was adjusted to 5 and the extraction process was performed at a temperature of 45 °C. Under these conditions, a pre-concentration factor of 25 was achieved and the total capacity of the stir bar for copper uptake was found to be 1200 μg. A non-imprinted polymer coating was used for comparison. The MIP stir bar showed sufficient mechanical and chemical stability and was able to perform more than 50 extractions without any damage to the polymer phase. The reproducibility between stir bars (n = 5) was 8.9%. The detection limit of the proposed method was found to be 0.38 μg L−1 with a relative standard deviation within 0.56–5.3% and a dynamic range between 1 and 1000 μg L−1. The method was successfully applied to the pre-concentration and determination of copper in a few different water samples.

A sensitive and specific high-performance liquid chromatography-tandem mass method (LC-MS/MS) has been developed and validated for the simultaneous determination of baicalin, chlorogenic acid and forsythin in dog plasma using a chemical synthetic compound buspirone as the internal standard (IS). Chromatographic separation was accomplished on a Phenomenex Gemini C18 column (2.0 × 50 mm, I.D., 3.5 μm) with a gradient elution system composed of 0.1% formic acid in water and 0.1% formic acid in methanol at a flow rate of 0.40 mL min−1. Detection was based on a triple quadrupole mass spectrometer using a multiple reaction monitoring (MRM) mode with an electrospray ionization source (ESI). The precursor/product transitions (m/z) in the positive ion mode were 447.3 → 271.1, 355.3 → 163.3, 556.9 → 309.3, and 386.5 → 122.1 for baicalin, chlorogenic acid, forsythin and IS, respectively. All of the calibration curves showed good linearity (r > 0.995) over the concentration range of 1.0 to 1000 ng mL−1 and the lower limits of quantification (LLOQs) were all 1.0 ng mL−1 for three analytes. The intra-day and inter-day precisions (% RSD) of low-level QC samples were all less than 20% and the precisions (% RSD) of high- and medium-level were all less than 15%. The accuracies at three QC levels ranged from 80 to 120%. The mean recoveries were all above 80% for three analytes. All the three analytes in dog plasma were stable at three different conditions. The well-established and fully validated method was firstly applied to the pharmacokinetics of baicalin, chlorogenic acid and forsythin in dogs after intravenous administration of Shuang-huang-lian freeze-dried powder. Significant double and/or multiple-peak phenomena in plasma concentration–time profiles of three analytes were observed in all six dogs, which are thought to be the results of the enterohepatic cycle.

Mercury emissions in equilibrium: a novel approach for the quantification of mercury emissions from contaminated soils by Manuel Carmona; Williams Llanos; Pablo Higueras; David Kocman (2793-2801).
Mercury emissions from soil samples with different mercury contents have been estimated using a closed circuit array. The samples were collected from the Almadén mercury mining district. The emissions confirmed that temperature and light radiation favour mercury desorption due to the increase in the mercury vapour pressure. An additional positive factor could be the photocatalytic reduction of soluble Hg2+ to volatile Hg0 at the soil surface. A physicochemical model based on mass transfer and equilibrium was developed and was used to reproduce the mercury emissions at the laboratory scale. The use of this model allowed us to obtain the unknown mass transfer coefficient (KL) and adsorption parameters required to quantify the possible gaseous mercury fluxes from these contaminated soils. Experimental results indicate that an equilibrium between the solid and gas phases was established. The proposed kinetic model reproduced perfectly the experimental data, with KL found to be proportional to the inverse of temperature and independent of the radiation. The concentration of mercury in the gas phase was mainly dependent on the soluble mercury content (HgS). Equilibrium data were fitted by Langmuir and Freundlich models and the best fit was obtained using the multi-layer model attending to the convex shape of the curves, which is characteristic of non-porous or possibly macroporous materials having a low adsorption energy. The Freundlich constant (KF) was also fitted as a polynomial function with temperature and this gave a straight line for the light radiation and a second grade equation for dark conditions. Once the parameters had been obtained, the Hg emission fluxes from contaminated soils were estimated and the values were between two and three orders of magnitude higher than those published in the literature for non-contaminated soils.

We report a simple analysis method for some common saccharides in aerosol samples on a diol-silica column, in connection with high performance liquid chromatography-evaporative light scattering detection. Under the established chromatographic conditions, the saccharides exhibit typical hydrophilic interaction behaviour with the stationary phase. Prior to chromatographic separation, the effectiveness of titania was studied as a specific trapping material toward saccharides in atmospheric samples. This approach simplifies the sample preconcentration and clean-up rendering the method fast and effective. For convenience during the isolation, magnetic particles were coated with a layer of titania. The actual LOD and LOQ values of the method range from 0.30 to 1.50 μg mL−1 and 1.00 to 5.00 μg mL−1, respectively, for the selected saccharides. The within-day repeatability (n = 3) was found to be less than 1.4% for concentrations in the range of 1.0–50.0 μg mL−1. An aerosol sample originating from the city centre of Ioannina was analyzed to verify the applicability of the method. Most of the saccharides were detected at concentrations from 83 ng m−3 (mannitol) to 200 ng m−3 (mannosan and levoglucosan) after the sample had been subjected to the preconcentration/clean-up procedure.

A novel graphene grafted silica-coated Fe3O4 (Fe3O4@SiO2-G) was synthesized and used as an efficient magnetic solid-phase extraction adsorbent for the simultaneous extraction of four neonicotinoid pesticides (thiamethoxam, imidacloprid, acetamiprid and thiacloprid) from pear and tomato samples followed by high performance liquid chromatography-diode array detection. After extraction, the adsorbent could be conveniently separated from the sample solution by an external magnetic field. The main experimental parameters affecting the extraction efficiency were investigated in detail. Under the optimum conditions, a rapid and effective determination of the neonicotinoid pesticides in pear and tomato samples was achieved. The limits of detection (S/N = 3) of the method for the neonicotinoids ranged from 0.08 to 0.15 ng g−1. Good linearity and reproducibility were obtained. The recoveries of the method were in the range from 93.1% to 107.4%.

Direct analysis of urinary 1-hydroxypyrene using extractive electrospray ionization ion trap tandem mass spectrometry by Xue Li; Xiaowei Fang; Zhiqiang Yu; Guoying Sheng; Minghong Wu; Jiamo Fu; Huanwen Chen (2816-2821).
Fast detection (0.5 min) of 1-hydroxypyrene (1-OHP) in urine and hydrolyzed urine without pre-treatment has been successfully achieved by using extractive electrospray ionization ion trap tandem mass spectrometry (EESI-MS/MS) under optimized EESI and MS/MS conditions. Experimental results indicated that for MS/MS analysis the operating parameter of activation Q (AQ) was critical for 1-OHP fragmentation in collision induced dissociation (CID) experiments, and in the EESI process the primary ESI solvent was a key factor for extractive ionization of urinary 1-OHP. The limit of detection (LOD) and limit of quantification (LOQ) were 0.75 and 2.25 μM for both urine and hydrolyzed urine samples. A five-point working curve ranging from 2.29 to 22.91 μM for 1-OHP in urine or hydrolyzed urine was obtained (R2 = 0.9941 for urine and R2 = 0.9983 for hydrolyzed urine), and the relative standard deviations (RSD, n = 6) were 2.6–9.7% and 1.5–6.4%, respectively. The developed EESI-MS/MS method was validated by detecting 1-OHP in both urine and hydrolyzed urine samples. Recoveries were determined to >50% and 0.5 min was taken for each measurement, indicating that the proposed method is a promising strategy for high throughput analysis of urinary 1-OHP required for health risk assessment of exposure to polycyclic aromatic hydrocarbons (PAHs).

Development of a HPLC-ECD method for the simultaneous determination of three synthetic estrogens in milk by Xiaojing Du; Yan Wu; Han Zhou; Ni Cheng; Mengxue Feng; Jianbin Zheng; Wei Cao (2822-2826).
A simple, accurate and inexpensive high performance liquid chromatography with electrochemical detection (HPLC-ECD) method for the simultaneous separation and determination of the three synthetic estrogens including diethylstilbestrol, dienestrol, and hexestrol in milk was established. For the sample treatment procedure, acetonitrile was added to the milk sample, the mixture was vortex mixed, centrifuged, concentrated by evaporation and determined by HPLC-ECD. The detection and quantification limits of the three estrogens obtained with ECD were lower than those obtained by diode array detection (DAD). Calibration curves, limit of detection and recoveries of diethylstilbestrol, dienestrol, and hexestrol with ECD ranged from 0.04 to 24 μg ml−1, 4.7 × 10−3 to 7.1 × 10−3μg ml−1, and 83 to 103%, respectively. Meanwhile, the effect of thermal treatment on the stability of the estrogens was investigated, and the results showed that these compounds initially decreased by 26% for diethylstilbestrol, 32% for dienestrol, and 41% for hexestrol after five minutes of heat treatment, then remained stable with increasing duration of heat treatment. The method was applied to the determination of the three synthetic estrogens in authentic milk samples.

Migration of 16 phthalic acid esters from plastic drug packaging to drugs by GC-MS by Lang Ji; Qing Liao; Lian Wu; Wei Lv; Ming Yang; Li Wan (2827-2834).
Phthalic acid esters (PAEs), which are human endocrine disruptors, can lead to sperm production atrophy and decreased sperm count and are poisonous to the embryo and active as anti-androgens. Many studies have been conducted to determine PAE levels in drugs and their corresponding plastic packaging; however, there is no systematic research on the transfer of PAE from plastic packaging to drugs. In this study, a simple and convenient method using GC-MS was developed for the simultaneous determination of 16 PAEs. n-Hexane was used to extract PAEs from the samples, and the extract was obtained by primary and secondary amine (PSA) and Florisil clean-up. The different parameters affecting both stages were carefully optimized. Subsequently, a method was developed that proved to be accurate and precise for the detection of 16 phthalates. This method was successfully applied to study PAE migration. The results showed that concentrations of dibutyl phthalate, diisobutyl phthalate and bis (2-ethylhexyl) phthalate in samples increased as a function of storage time. A positive correlation between the amount of PAEs that migrated and storage time during the entire migration process was confirmed. The PAE levels did not exceed the legal limits established by the European Union (0.3 ppm for food); however, changes in drug ingredients and excipients, as well as other factors, may lead to increased PAE migration. Therefore, this phenomenon should receive further attention.

A novel high throughput nucleic acid sandwich hybridization assay on a gold substrate by C. H. van den Kieboom; T. S. Y. van Domburg; M. I. de Jonge; G. Ferwerda; P. W. M. Hermans (2835-2839).
Biosensors will become increasingly important for medical diagnostics. An essential part of biosensor development is assay optimization; to find the optimal conditions a large number of variables need to be tested and high throughput online measurements are time-consuming or sometimes impossible. In this study, an offline high throughput nucleic acid sandwich hybridization assay is developed for pre-screening assay conditions on a gold substrate. The assay is performed in a 48-well plate using alkanethiolated ssDNA molecules, which bind to the substrate surface through a specific thiol–gold interaction and function as the recognition site for target DNA. The hybridization reaction was analyzed using an enzymatic read-out system measuring DNA–DNA interactions in a highly specific and sensitive manner, since only fully complementary nucleotide sequences were detected with a lower limit of detection of 10 pmol l−1. This high throughput low cost offline method has been demonstrated to be flexible and is expected to improve and accelerate biosensor development.

Recently, there was much attraction in amphiphilic block copolymers composed of methoxy poly(ethylene glycol) (MePEG) and biodegradable polyesters such as poly(caprolactone) (PCL). This copolymer significantly has applications in drug delivery systems. According to literature reports, efficiency of drug delivery and particle cellular uptake could be affected by many characteristics, such as particle size, different compositions of the particles, surface properties, different cell lines and cell densities, temperature. Hu et al. showed that the particle size is the key factor to determine the cellular uptake efficiency and it is found that the composition and molar mass of the copolymers are influential on the particle size. The present paper firstly discusses response surface methodology (RSM) as an efficient system for building a mathematical model and optimization of the apparent molar mass (Mn) by size-exclusion chromatography (SEC) (Y1) and nuclear magnetic resonance (1H NMR) (Y2). In this work, RSM is used to optimize the physical and chemical variables such as time, temperature, MePEG/CL and amount of Sn(Oct)2. A 24 small factorial central composite design (CCD) was employed. After evaluation of experimental results, a 2F1 model was found to be fit for Y1 response (SEC) and quadratic model for Y2 (NMR) without any requirement of power transformation. The selection of model was done on basis of high adjusted R2 value and lowered p-value for each model in individual analysis of each response. Through desirability analysis, it was found that SEC has a good precision/repeatability but a generally poor accuracy/reproducibility in determination of Mn. NMR is not suitable for determination of Mn for high molar mass PEG–PCL copolymers. Thus, the information that can be derived from NMR spectra is restricted because of broad and overlapping signals, poor solubility of polymers or non-availability of suitable solvents. NMR coupled with partial least squares (PLS) regression is applied to determine the Mn of MePEC in the present study. A PLS regression model with a coefficient of determination (R2) of 0.8444 and root-mean-square error of cross validation (RMSECV) of 0.5625 was obtained in the spectral range δ 0.28–7.28 ppm. The results obtained in this study indicate that 1H NMR spectroscopy can be used as an easy, rapid and novel tool to quantitatively predict the Mn of MePEC.

The sensitive determination of clenbuterol (CLB) and salbutamol (SAL) in swine urine was developed by using capillary electrophoresis with a moving reaction boundary (MRB)-based stacking method. Under the optimum conditions, the MRB-based stacking procedure produced an improved concentration sensitivity of 70.5-fold for CLB and 24.7-fold for SAL. The improvement results in a limit of detection (LOD) of about 0.26 ng mL−1 and 0.96 ng mL−1 for CLB and SAL, respectively. The method has been successfully used for the analysis of CLB and SAL in swine urine, and the R.S.D. was less than 5.0%, the recoveries were in the range of 96.8–103.6% and the linear ranges of CLB and SAL were 0.003–10.0 μg mL−1 and 0.01–20.0 μg mL−1, respectively.

Determination of polyamines in Arabidopsis thaliana by capillary electrophoresis using salicylaldehyde-5-sulfonate as a derivatizing reagent by Genki Inoue; Takashi Kaneta; Toshio Takayanagi; Junichi Kakehi; Hiroyasu Motose; Taku Takahashi (2854-2859).
Herein, we report a novel method for the determination of polyamines in a sample extracted from Arabidopsis thaliana by capillary electrophoresis (CE) using salicylaldehyde-5-sulfonate (SAS) as a derivatizing reagent. An aldehyde group of SAS forms a Schiff base with amino groups of aliphatic polyamines, resulting in an anionic species with an absorption band in the ultraviolet region. The derivatization method was straightforward since the derivatives were formed by mixing a sample with the derivatizing reagent at a neutral pH. In addition, the negative charges induced by SAS led to a high resolution with a short analysis time. This method permitted the separation of five polyamines, which play important roles in plants. However, further improvement in sensitivity was needed for the determination of the polyamines in plant samples. Therefore, the CE method was coupled with solid-phase extraction (SPE) using an ion-pairing formation with sodium dodecyl benzene sulfonate. The SPE method improved the concentration limits of detection to sub-μM levels, which corresponded with a 10-fold enhancement. The calibration curves for cadaverine, putrescine, and spermidine were linear with concentrations that ranged from 1 to 20 μM and correlation coefficients (R2) were greater than 0.998. The proposed method was applied to the determination of spermidine in a plant sample, Arabidopsis thaliana.

Hydrolysis of lysozyme with an RF-powered micro-reactor by Valerie J. Scott; Peter H. Siegel; Xenia Amashukeli (2860-2865).
An RF sample-processing micro-reactor that was developed as part of potential in situ exploration missions to inner- and outer-planetary bodies has been used to perform hydrolysis of a protein—lysozyme. The micro-reactor was designed to utilize aqueous solutions subjected to 60 GHz radiation at 730 mW input power to extract target organic compounds and molecular and inorganic ions as well as to hydrolyze complex polymeric materials. High performance liquid chromatography (HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used in the analysis of the reaction products. It was established that both the flow rate of the protein solution through the reactor (2.5–87 μL min−1) and the applied head pressure (0.34–2.76 MPa) positively affect the hydrolysis reaction when exposed to RF radiation. The results of the RF micro-reactor samples were compared to those run in an analogous sample handling setup using a heat source in place of RF radiation.

We have fabricated a sensitive acetylcholinesterase biosensor that is based on dual-signal amplification. A large amount of enzyme was immobilized on a glassy carbon electrode via specific binding between functionalized ZnSe quantum dots and acetylcholinesterase, and a graphene–chitosan nanocomposite was introduced that improves the response. These two factors render the biosensor highly sensitive to acetylthiocholine chloride. Organophosphate pesticides were detected with this biosensor using methyl parathion as a model enzyme inhibitor. Under optimal conditions, there is a linear relationship between the percentage of inhibition (I%) and the log of the concentration of methyl parathion in the 0.5 nM to 0.5 μM range, with a 0.2 nM detection limit (at an S/N of 3). The biosensor displays acceptable reproducibility and relatively good storage stability. It was successfully employed to the determination of methyl parathion in spiked water and soil samples.

We have constructed a nanoconjugate composed of positively charged NaYF4:Yb3+,Er3+ upconversion nanoparticles (UCNPs) and negatively charged gold nanoparticles (AuNPs). Fluorescence resonance energy transfer occurs between the UCNPs and the AuNPs. If the AuNPs interact with thiols, this will cause aggregation and lead to the decreased absorption by the AuNPs. As a result, the luminescence intensity of the UCNPs will be restored. This finding was explored to design a simple and sensitive luminescence assay for biothiols including cysteine, homocysteine. Under the optimal conditions, the calibration plot is linear in the range from 0.04 to 1.00 μg mL−1 of cysteine, and from 0.06 to 1.80 μg mL−1 of homocysteine. This method was applied to the determination of cysteine in synthetic samples.

Hydroxylated polymethoxyflavonoid glycosides (OH-PMFGs) are a rare kind of highly methoxylated flavone glycosides, which demonstrate many health-promoting bioactivities. Here we aim to identify OH-PMFGs from the leaves of Murraya paniculata using EIC (extracted ion chromatogram)-MS coupled with diagnostic product ions (DPIs) analytical method. Respective DPIs for glycosides of polymethoxyflavones, polymethoxyflavanones and polymethoxychalcones were obtained from the fragmentation pathways of eleven PMF (polymethoxylated flavonoid) standards. A sensitive HPLC-DAD-ESI-MS/MS was established for simultaneous qualitative and quantitative determination of the main OH-PMFGs in the extract. 54 OH-PMFGs including 49 flavone glycosides and 5 flavanone or chalcone glycosides were screened and identified from the leaves of M. paniculata. Meanwhile, the contents of three main OH-PMFGs in the extract were determined by HPLC-UV. It was the first systematic report of the presence of rare OH-PMFGs in the plants of the genus Murraya. The results indicated that the methodology developed could be employed as an effective technique for structural characterization of OH-PMFGs in complex extracts of TCMs (traditional Chinese medicines).

Ultrafiltration to remove trypsin for suppressing the back-exchange of 18O labeling by Yan Xiong; Yujuan Li; Kefu Liu; Ming Ke; Umer Awan; Yulin Deng (2892-2897).
The post-digestion of 18O labeling is widely used in comparative proteomics for the relative quantification of proteins and peptides. Application and precise quantitative analysis are hindered, since 18O labeling is pH sensitive and has back-exchange. Herein, in a study of peptides derived from BSA (bovine serum albumin), we demonstrated through a detailed evaluation that removal of soluble trypsin by ultrafiltration prevented back-exchange and effectively enhanced the stability of 18O-labeled peptides under off-gel separation even without trypsin inhibitor. After ultrafiltration, the 18O labeling efficiency was 95.8 ± 2.3% under off-gel separation. In addition, these peptides had a relative high, approximately 80% recovery and less than 5% 16O/18O ratio variation through ultrafiltration, indicating no apparent effect on quantification. Hence, the useful and economical method presented here effectively inhibited back-exchange in off-gel separation and might enable further applications towards large-scale biomarker discovery.

Back cover (2899-2900).