Analytica Chimica Acta (v.631, #1)

Mercury in contaminated soils and sediments could be extracted by various chemical reagents in order to determine the different mercury species and partitions, providing useful information of toxicology, bioavailability and biogeochemical reactivity. Unfortunately, at present, neither specific extractants nor standard protocols exist for the isolation of particular mercury species. Although there has been considerable research focused on reagents for extracting mercury species, there is still little consensus. Thus, workers are advised to select the most appropriate reagent based on the nature of their sample, and to take all possible steps to validate the analyses performed. Therefore, the aim of this paper is to review the current reagents used for determining total mercury and its speciation as well as fractionation such as methylmercury, ethylmercury, elemental mercury, mercury sulphide and organically bound mercury by supposed selective (one reagent) and sequential (several reagents) extractions. The gathering information presented here bring to light the need for standard protocol for which the used chemical reagents should take into account the particular chemistry of mercury associated with specific properties of soil and sediment. Beside this required scheme, appropriate reference materials are also demanded.
Keywords: Mercury speciation; Chemical extraction; Reagents; Soils; Sediments;

This paper presents a Bayesian approach to the development of spectroscopic calibration models. By formulating the linear regression in a probabilistic framework, a Bayesian linear regression model is derived, and a specific optimization method, i.e. Bayesian evidence approximation, is utilized to estimate the model “hyper-parameters”. The relation of the proposed approach to the calibration models in the literature is discussed, including ridge regression and Gaussian process model. The Bayesian model may be modified for the calibration of multivariate response variables. Furthermore, a variable selection strategy is implemented within the Bayesian framework, the motivation being that the predictive performance may be improved by selecting a subset of the most informative spectral variables. The Bayesian calibration models are applied to two spectroscopic data sets, and they demonstrate improved prediction results in comparison with the benchmark method of partial least squares.
Keywords: Bayesian inference; Multivariate calibration; Multivariate linear regression; Partial least squares; Variable selection;

An integration strategy to estimate the initial rates of enzyme reactions with much expanded linear ranges using uricases as models by Beizhong Liu; Yunsheng Zhao; Lina Zhao; Yanlin Xie; Sha Zhu; Zhirong Li; Yin Liu; Wei Lu; Xiaolan Yang; Guoming Xie; Huansi Zhong; Ming’an Yu; Hong Liao; Fei Liao (22-28).
A new strategy was proposed to estimate the initial rates of reactions catalyzed by Michaelis–Menten enzymes via integrating the classical initial rate method for low activities with an improved integrated method for high activities. Between these two individual methods, this integration strategy required: (a) the consistent linear response slopes, acquired with an optimized preset substrate concentration (PSC) to derive the initial rates from the maximal reaction rates estimated by the improved integrated method; (b) an overlapped region of the initial rates measurable with consistent results, realized with an optimized reaction duration to record reaction curves for analyses by the improved integrated method; (c) a switch cutoff, preset as the instantaneous substrate concentration slightly above that after a given lag time when the enzyme activity was just below the upper limit for the linear response of the classical initial rates. By simulation with uricases at a given initial substrate concentration (S 0), the optimized PSC was 93% S 0, the optimized reaction duration at S 0 from 0.35-fold to 11.0-fold Michaelis–Menten constant (K m) was within 6.0 min and the switch cutoff was available at the given S 0 after 30-s lag time, all of which were combined to produce 300-fold linear ranges. By experimentation with one uricase of K m at 6.6 μM and the other uricase of K m at 220 μM under optimized conditions, this integration strategy with S 0 at 75 μM produced 100-fold linear ranges. Thus, this integration strategy exhibited much expanded linear ranges and practical efficiency over wide ratios between S 0 and K m.
Keywords: An integration strategy; Enzyme activity; Initial rates; Linear ranges; The classical initial rate method; The improved integrated method;

Validated quantitative structure–activity relationship analysis of a series of 2-aminothiazole based p56Lck inhibitors by Jiazhong Li; Juan Du; Lili Xi; Huanxiang Liu; Xiaojun Yao; Mancang Liu (29-39).
In this study, the quantitative structure–activity relationship (QSAR) of a series of 2-aminothiazole based Lck inhibitors was investigated. The key structural features responsible for the inhibition activities were discussed in detail. A population of 100 rigorously validated linear QSAR models were established based on the descriptors calculated in DRAGON program and selected by genetic algorithm (GA). A total of 36 descriptors were involved in all the QSAR models. Then the common descriptors appeared in all the models were extracted to build the final QSAR model. As a result, the final 8-parameter QSAR model was established. After analysis of the eight descriptors, some advice was proposed to help the design of possible novel inhibitors with higher bioactivity.
Keywords: Lymphocyte-specific kinase (LCK); Genetic algorithm (GA); Multiple linear regression (MLR); Quantitative structure–activity relationship (QSAR);

In this work, electrochemical methods were used to prepare complexes with Au and Cl species on bulk Au substrates. Then the electrochemically roughened Au substrates were further heat-treated at different temperatures. The effect of temperatures used in heat treatments between 25 and 100 °C on electrocatalytical polymerization of polypyrrole (PPy) formed on the prepared gold substrates was first investigated. The result indicates that the optimally electrocatalytical capability of the heat-treated Au substrate for PPy polymerization is at 75 °C. Moreover, the autopolymerized PPy on the roughened Au substrate treated at 75 °C demonstrates the highest oxidation level and oxidation degree of 0.32 and 0.50, respectively. Primary results indicate that complexes with positively charged Au act as oxidants, and perchlorate and chloride ions act as dopants for the oxidation-polymerization of PPy.
Keywords: Temperature; Electrocatalytical polymerization; Gold; Polypyrrole;

A kind of homemade solid-phase microextraction fibre coupled with gas chromatography–mass spectrometry (GC–MS) was developed for trace analysis of antiestrogens (tamoxifen, cis- and trans-clomiphene) in biological matrices. In this method, derivatization was unnecessary and sample solution could be injected directly after very simple deproteinization operation. The conditions of influencing adsorption of the solid-phase microextraction (SPME) fibre and desorption of the analytes were investigated in details. Matrix effects were studied in different background. Under optimum conditions, the proposed method was further validated by spiking analytes into rabbit liver solutions. Linear ranges of tamoxifen, cis- and trans-clomiphene were 0.02–2.56, 0.08–2.56 and 0.16–2.56 ng mL−1, respectively. The limits of quantitation were in the range of 0.02–0.16 ng mL−1. The intra-day accuracy was ranged 96.2–106.2% and precision were in the range of 5.1–8.7%. The extraction recoveries of the antiestrogens in rabbit liver solution were between 73.8% and 113.1%, and R.S.D.s were from 3.6% to 14.1%. The results show that the homemade sol–gel coating is suitable for determination of trace antiestrogens in complex matrices. The proposed approach was proved to be rapid, simple, easy, sensitive and reproducible for trace analysis of antiestrogens in biological matrices.
Keywords: Solid-phase microextraction; Antiestrogens; Gas chromatography–mass spectrometry; Biological matrices;

In this study, a new sampling method called headspace mulberry paper bag micro solid phase extraction (HS-MPB-μ-SPE) combined to gas chromatography–mass spectrometry has been applied for the analysis of volatile aromas of liquid essential oils from Bulgarian rose and Provence lavender. The technique uses an adsorbent (Tenax TA) contained in a mulberry paper bag, minimal amount of organic solvent. Linearities for the six-points calibration curves were excellent. LOD values were in the rage from 0.38 ng mL−1 to 0.77 ng mL−1. Overall, precision and recovery were generally good. Phenethyl alcohol and citronellol were the main components in the essential oil from Bulgarian rose. Linalyl acetate and linalool were the most abundant components in the essential oils from true lavender or lavandin. Additionally, the relative extraction efficiencies of proposed method have been compared with HS-SPME. The overall extraction efficiency was evaluated by the relative concentration factors (CF) of the several characteristic components. CF values by HS-MPB-μ-SPE were lower than those by headspace solid phase microextraction (HS-SPME). The HS-MPB-μ-SPE method is very simple to use, inexpensive, rapid, requires small sample amounts and solvent consumption. In addition, this method allowed combining of extraction, enrichment, and clean-up in a single step. HS-MPB-μ-SPE and GC/MS is a promising technique for the characterization of volatile aroma compounds from liquid essential oils.
Keywords: Mulberry paper bag; Micro solid phase extraction; Gas chromatography/mass spectrometry; Aroma; Bulgarian rose essential oil; Provence lavender essential oil;

Low-temperature nonthermal plasma has been used to prepare solid-phase microextraction (SPME) fibers with high adsorbability, long-term serviceability, and high reproducibility. Graphite rods serving as fiber precursors were treated by an air plasma discharged at 15.2–15.5 kV for a duration of 8 min. Sampling results revealed that the adsorptive capacity of the homemade fiber was 2.5–34.6 times that of a polyacrylate (PA) fiber for alcohols (methanol, ethanol, isopropyl alcohol, n-butyl alcohol), and about 1.4–1.6 times and 2.5–5.1 times that of an activated carbon fiber (ACF) for alcohols and BTEX (benzene, toluene, ethylbenzene, and xylenes), respectively. It is confirmed from FTIR (Fourier transform infrared spectrophotometer) and SEM (scanning electron microscope) analyses that the improvement in the adsorptive performance attributed to increased surface energy and roughness of the graphite fiber. Using gas chromatography (GC)-flame-ionization detector (FID), the limits of detection (LODs) of the alcohols and BTEX ranged between 0.19 and 3.75 μg L−1, the linear ranges were between 0.6 and 35619 μg L−1 with good linearity (R 2  = 0.9964–0.9997). It was demonstrated that nonthermal plasma offers a fast and simple method for preparing an efficient graphite SPME fiber, and that SPME using the homemade fiber represents a sensitive and selective extraction method for the analysis of a wide range of organic compounds.
Keywords: Solid-phase microextraction; Graphite fiber; Nonthermal plasma; Adsorption;

Evaluation of sorption of uranium onto metakaolin using X-ray photoelectron and Raman spectroscopies by Jamil R. Memon; Keith R. Hallam; Muhammad I. Bhanger; Adel El-Turki; Geoffrey C. Allen (69-73).
Metakaolin prepared from a natural clay mineral ore of aluminium kaolinite is a promising low cost and high activity aluminosilicate material that has been investigated for studying the sorption behavior of uranium. Here, metakaolin was characterized using X-ray photoelectron spectroscopy (XPS) and the effects of pH, contact time and initial metal ion concentration on its sorption behavior were studied. The sorption process was found to initially be rapid (∼60% at time 0 min) but became slower with time; equilibrium was established within 24 h (∼80% sorption). The data were applied to study the kinetics of the sorption process. The Langmuir and Dubinin–Radushkevich (D–R) sorption isotherms were used to describe partitioning behavior for the system at room temperature. The binding of metal ions was found to be pH dependent, with optimal sorption occurring at pH 5. The retained metal ions were eluted with 5 mL of 0.1 M HNO3. Raman spectroscopy and XPS were used to evaluate the sorption mechanism of U(VI).
Keywords: Metakaolin; Uranium; Sorption; Kinetics; Isotherms;

Lanthanum hydroxide precipitate is for the first time coated onto cellulose fibre and serves as a novel sorption medium for separation and speciation of inorganic selenium. A micro-column packed with precipitate-layer-coated cellulose fibre is incorporated into a sequential injection system for selenite retention from a neutral aqueous solution, which is afterwards stripped with a NaBH4–NaOH solution as eluent. The hydride generation is actuated by merging the eluate and hydrochloric acid downstream, followed by the detection with atomic fluorescence spectrometry. Total inorganic selenium is derived by pre-reduction of selenate and speciation is estimated by difference. The coated precipitate layer can be used for 150 runs for selenium sorption, offering a clear advantage over the conventional precipitation protocols where a large amount of precipitate is dissolved into a small volume of eluent which might interfere with the detection. With a sample volume of 1.0 mL, an enrichment factor of 9.7 and a detection limit of 9 ng L−1 are obtained in a linear range of 0.05–2.5 μg L−1. A sampling frequency of 24 h−1 is achieved along with a R.S.D. of 1.7% at 0.5 μg L−1 Se(IV). The procedure is validated by analyzing selenium in a reference material GBW 10010 (rice) and a human hair sample. It is further demonstrated by speciation of inorganic selenium in surface water samples by pre-reduction of selenate.
Keywords: Precipitate layer coating; Lanthanum hydroxide; Selenium preconcentration and speciation; Sequential injection; Hydride generation atomic fluorescence spectrometry;

Determination of endogenous steroids in complex matrices such as cattle's meat is a challenging task. Since endogenous steroids always exist in animal tissues, no analyte-free matrices for constructing the standard calibration line will be available, which is crucial for accurate quantification specially at trace level. Although some methods have been proposed to solve the problem, none has offered a complete solution. To this aim, a new quantification strategy was developed in this study, which is named “surrogate analyte approach” and is based on using isotope-labeled standards instead of natural form of endogenous steroids for preparing the calibration line. In comparison with the other methods, which are currently in use for the quantitation of endogenous steroids, this approach provides improved simplicity and speed for analysis on a routine basis. The accuracy of this method is better than other methods at low concentration and comparable to the standard addition at medium and high concentrations. The method was also found to be valid according to the ICH criteria for bioanalytical methods. The developed method could be a promising approach in the field of compounds residue analysis.
Keywords: Endogenous steroids; Surrogate analyte; Isotope-labeled standard; Meat; Gas chromatography mass spectrometry;

Arsenic pollution of public water supplies has been reported in various regions of the world. Recently, some cancer patients are treated with arsenite (AsIII); most Japanese people consume seafoods containing large amounts of negligibly toxic arsenic compounds. Some of these arsenic species are metabolized, but some remain intact. For the determination of toxic AsIII, a simple, rapid and sensitive method has been developed using electrospray ionization mass spectrometry (ESI-MS). AsIII was reacted with a chelating agent, pyrrolidinedithiocarbamate (PDC, C4H8NCSS) and tripyrrolidinedithiocarbamate-arsine, As(PDC)3, extracted with methyl isobutyl ketone (MIBK). A 1 μL aliquot of MIBK layer was directly injected into ESI-MS instrument without chromatographic separation, and was detected within 1 min. Arsenate (AsV) was reduced to AsIII with thiosulfate, and then the total inorganic As was quantified as AsIII. This method was validated for the analysis of urine samples. The limit of detection of As was 0.22 μg L−1 using 10 μL of sample solution, and it is far below the permissible limit of As in drinking water, 10 μg L−1, recommended by the WHO. Results were obtained in < 10 min with a linear calibration range of 1–100 μg L−1. Several organic arsenic compounds in urine did not interfere with AsIII detection, and the inorganic As in the reference materials SRM 2670a and 1643e were quantified after the reduction of AsV to AsIII.
Keywords: Arsenite; Arsenate; Mass spectrometry; Electrospray ionization; Pyrrolidinedithiocarbamate;

A highly sensitive chromo- and fluorogenic chemodosimeter for sulfide anion was developed based on its nucleophilicity. 2,4-Dinitrobenzenesulfonyl-fluorescein (I) is a weakly fluorescent compound. Upon mixing with sulfide anion in aqueous acetone solution, the 2,4-dinitrobenzenesulfonyl group of I was efficiently removed and highly fluorescent fluorescein was released, hence leading to the dramatic increases in both fluorescence and absorbance of the reaction solution. The fluorescence increment is linear with sulfide anion concentration in the range 50–1000 nmol L−1 with a detection limit of 4.3 nmol L−1 (3σ). The proposed chemodosimeter showed excellent selectivity toward sulfide anion and was successfully applied to the determination of sulfide anion in synthetic wastewater samples.
Keywords: Chemodosimeter; Fluorescein; Sulfide anion; Fluorescence;

A labeling-free surface plasmon resonance (SPR) sensor technique was used to monitor the conformational changes of immobilized globular proteins (RNase A and Lysozyme) in chemical unfolding and refolding. The conformational changes of proteins at solid/liquid interface are characterized as two-state transformation (S-shaped) curves through matrix-effect correction and theoretic estimation. By extrapolation with a Santoro–Bolen equation, the SPR results for both reductive immobilized proteins are estimated to 1.9 kcal mole−1 global free energy (ΔG U) in urea-induced unfolding. But the ΔG U for RNase A and Lysozyme in GdmCl-induced unfolding are 1.5 and 2.15 kcal mole−1, respectively. The disagreement in free energy is partially accounted for by the differences of intra-molecular interactions and immobilization.
Keywords: Surface plasmon resonance; Conformation; Unfolding; Immobilization; Disulfide bond;

Kinetic capillary electrophoresis-based affinity screening of aptamer clones by Diana Yunusov; Mandy So; Solmaz Shayan; Victor Okhonin; Michael U. Musheev; Maxim V. Berezovski; Sergey N. Krylov (102-107).
DNA aptamers are single stranded DNA (ssDNA) molecules artificially selected from random-sequence DNA libraries for their specific binding to a certain target. DNA aptamers have a number of advantages over antibodies and promise to replace them in both diagnostic and therapeutic applications. The development of DNA aptamers involves three major stages: library enrichment, obtaining individual DNA clones, and the affinity screening of the clones. The purpose of the screening is to obtain the nucleotide sequences of aptamers and the binding parameters of their interaction with the target. Highly efficient approaches have been recently developed for the first two stages, while the third stage remained the rate-limiting one. Here, we introduce a new method for affinity screening of individual DNA aptamer clones. The proposed method amalgamates: (i) aptamer amplification by asymmetric PCR (PCR with a primer ratio different from unity), (ii) analysis of aptamer–target interaction, combining in-capillary mixing of reactants by transverse diffusion of laminar flow profiles (TDLFP) and affinity analysis using kinetic capillary electrophoresis (KCE), and (iii) sequencing of only aptamers with satisfying binding parameters. For the first time we showed that aptamer clones can be directly used in TDLFP/KCE-based affinity analysis without an additional purification step after asymmetric PCR amplification. We also demonstrated that mathematical modeling of TDLFP-based mixing allows for the determination of K d values for the in-capillary reaction of an aptamer and a target and that the obtained K d values can be used for the accurate affinity ranking of aptamers. The proposed method does not require the knowledge of aptamer sequences before screening, avoids lengthy (3–5 h) purification steps of aptamer clones, and minimizes reagent consumption to nanoliters.
Keywords: Affinity screening of aptamers; Kinetic capillary electrophoresis; Non-equilibrium capillary electrophoresis of equilibrium mixtures; Transverse diffusion of laminar flow profiles; Asymmetric polymerase chain reaction; MutS protein; SELEX;

Approaches for application of sub and supercritical fluid extraction for quantification of orbifloxacin from plasma and milk: Application to disposition kinetics by A.M. Abd El-Aty; Jeong-Heui Choi; Ma-Wou Ko; Sathya Khay; Ayman Goudah; Ho-Chul Shin; Jin-Suk Kim; Byung-Joon Chang; Chi-Ho Lee; Jae-Han Shim (108-115).
Since its extensive development in the early 1980s, SFE has attracted considerable attention as a sample-preparation procedure. However, other different sample preparation procedures, including precipitation, liquid- and/or solid-phase extraction in biological fluids, also remain in use. In this investigation, SFE was introduced to isolate and identify orbifloxacin from plasma and milk. Four parameters, including the temperature and the pressure of supercritical fluid, modifier ratios, and dynamic extraction time, were evaluated and optimized to obtain the best yield of the analyte from the biological fluids. Determinations of the orbifloxacin (OBFX) in the extracts were carried out using HPLC-FLD. The optimum conditions of the extraction process that yielded the maximum analyte extraction efficiencies were 150 °C vs. 60 °C, 250 kg cm−2, 30% vs. 35% methanol, and 40 min vs. 20 min, for plasma and milk, respectively. The linearity of the calibration curves as well as the instrument LODs/LOQs were evaluated. Good linearity (at least r 2  ≥ 0.999) of the calibration curves was obtained over the range from 0.2 to 0.01 μg mL−1. The method showed a good recovery rate (74.2–127.73%) and precision (RSDs: 1.64–20%). The instrumental LOD and LOQ values were 0.004 μg mL−1 vs. 0.01 μg mL−1 or 0.006 μg mL−1 vs. 0.02 μg mL−1, for plasma and milk, respectively. The method was successfully applied to estimate the pharmacokinetic variables of orbifloxacin in lactating does. To the best of our knowledge, this is the first time that SFE has been applied to isolate an antimicrobial agent from biological fluids. This method is promising for clinical applications and for pharmacokinetic studies of various pharmaceuticals in biological fluids.
Keywords: Biological fluids; Plasma; Milk; Small ruminants; Supercritical fluid extraction; Fluoroquinolones;

Development of sandwich enzyme-linked immunosorbent assay systems for plasma β-galactoside α2,6-sialyltransferase, a possible hepatic disease biomarker by Satoshi Futakawa; Shinobu Kitazume; Ritsuko Oka; Kazuko Ogawa; Yoshiaki Hagiwara; Akinori Kinoshita; Kazuya Miyashita; Yasuhiro Hashimoto (116-120).
Previous reports, including our work, have shown that plasma β-galactoside α2,6-sialyltransferase (ST6Gal I) activity is significantly increased in particular hepatopathological situations, suggesting that it may represent a sensitive biomarker for diagnosing hepatic diseases. So far, activity of ST6Gal I have been measured by using radioactive tracer method in place of measuring amount of ST6Gal I. However, this method is tangled and cannot exclude other sialyltransferase activities. Thus, simple and specific methods for measuring plasma ST6Gal I had been unavailable. Here, we developed two kinds of sandwich enzyme-linked immunosorbent assay (ELISA) systems that specifically detect the soluble cleaved form of ST6Gal I in plasma. In one sandwich ELISA, we detected rat specific sequence, EFQMPK, which is N-terminus of soluble ST6Gal I. In the other sandwich ELISA, we detected internal common sequence among rat, mouse and human ST6Gal I in plasma (M2 ELISA). Using the M2 ELISA, we observed that elevation of plasma ST6Gal I was much faster than elevation of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in a carbon tetrachloride (CCl4)-induced mouse liver injury model. Our data suggest that these ELISA systems are very useful tools for measuring plasma ST6Gal I, which represents a potential biomarker for diagnosing hepatic diseases.
Keywords: β-Galactoside α2,6-sialyltransferase; Enzyme-linked immunosorbent assay; Hepatic disease; Biomarker; Aspartate aminotransferase; Alanine aminotransferase;