Analytical Methods (v.6, #17)

Front cover (6591-6592).

Contents list (6593-6609).

Complex carbohydrates (glycans) play an important role in nature and study of their interaction with proteins or intact cells can be useful for understanding many physiological and pathological processes. Such interactions have been successfully interrogated in a highly parallel way using glycan microarrays, but this technique has some limitations. Thus, in recent years glycan biosensors in numerous progressive configurations have been developed offering distinct advantages compared to glycan microarrays. Thus, in this review advances achieved in the field of label-free glycan biosensors are discussed.

Kernel k-nearest neighbor classifier based on decision tree ensemble for SAR modeling analysis by Xin Huang; Qing-Song Xu; Dong-Sheng Cao; Yi-Ping Luo; Yi-Zeng Liang (6621-6627).
Kernel approaches that can effectively solve nonlinear problems using implicit nonlinear mapping have been gaining popularity in the field of chemistry. In the present study, a novel tree kernel k-nearest neighbor algorithm (TKk-NN) has been proposed. First, an informative novel tree kernel is constructed based on the decision tree ensemble. The constructed tree kernel can effectively use important variables for classification and neglect useless variables through variable importance ranking during the process of building the kernel. Under the framework of kernel methods, this tree kernel is then extended to the k-nearest neighbor algorithm. Three SAR datasets together with the simulated data were used to test the performance of k-NN with tree and radial basis function kernels. The results show that TKk-NN really is an attractive alternative technique.

A new screening method for recombinant Saccharomyces cerevisiae strains based on their xylose fermentation ability measured by near infrared spectroscopy by Hiroyuki Morita; Tomohisa Hasunuma; Maria Vassileva; Akihiko Kondo; Roumiana Tsenkova (6628-6634).
Fuel ethanol produced from lignocellulose by the yeast Saccharomyces cerevisiae becomes an increasingly important alternative to fossil fuels. Selection of S. cerevisiae strains, which can effectively produce ethanol from xylose is crucial to improve the fuel yield from lignocellulose. In the present study, a universal calibration system was developed by the combination of time series, fermentation near infrared (NIR) spectral data analysis and reference high-performance analysis of a single yeast strain which enabled the evaluation of the ethanol production ability of a wide variety of xylose-fermenting yeast strains. Subtraction of xylose and ethanol concentrations at 0 h for each clone, as well as the respective spectra, reduced subtle errors of the fermentation components naturally occurring in multiple experiments to clearly visualize the difference of fermentation ability between strains. Also, NIR spectra showed specific peaks in difference spectra calculated from the subtraction treatment. A robust univariate linear regression model led to high prediction accuracy of xylose consumption (R2 > 0.99) and ethanol production (R2 > 0.98) for a wide variety of yeast strains when using only the distinct spectral pattern of a single-strain. Here, a novel method for screening of high-performing yeast strains has been developed requiring only a simple single-strain calibration model. The advantages of NIR spectroscopy such as rapid and convenient experimental preparation using electromagnetic waves in the region, which provide deep penetration into an aqueous sample, are successfully exploited in the proposed screening method.

Detection of residual biocides in antibody drug conjugates for ImmunoPET imaging by Colin D. Medley; Jason Gruenhagen; Peter Yehl; Nik P. Chetwyn (6635-6640).
Antibody drug conjugates are comprised of monoclonal antibodies conjugated to a small molecule drug to combine the targeted delivery of the antibody with the mechanism of action of the small molecule component. One class of antibody drug conjugates is used in ImmunoPET and combines the selectivity of the antibody with a positron emission tomography imaging agent that enables imaging tumors based on binding of the antibody. As the positron emission tomography imaging reagents are limited by their radioactive half-life, they are prepared at a single patient GMP quality scale with limited advance notification of when a patient requires the dose. One aspect of this single batch manufacture is using PD-10 columns to purify the ImmunoPET agent from the excess radioactive material used in the manufacturing process. These columns are kept in a storage solution containing biocides, 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, which prevent microbial growth in the column but could also adversely affect the patient if injected. We have developed a validatable HPLC-UV/MS method for the detection of biocides in formulated ImmunoPET drug product samples and formulation buffer. This method enables the demonstration of the clearance of biocides in the columns prior to use and quantitation of any residual biocides present in the drug product.

In situ characterization by Raman and X-ray fluorescence spectroscopy of post-Paleolithic blackish pictographs exposed to the open air in Los Chaparros shelter (Albalate del Arzobispo, Teruel, Spain) by Àfrica Pitarch; Juan Francisco Ruiz; Silvia Fdez-Ortiz de Vallejuelo; Antonio Hernanz; Maite Maguregui; Juan Manuel Madariaga (6641-6650).
An in situ study of post-Palaeolithic blackish pictographs found in an open air rock-shelter, Los Chaparros site (Albalate del Arzobispo, Teruel province, Spain), was carried out to identify the black pigments used. The composition of the pigments was analyzed by means of non-invasive instrumentation, such as a portable Raman spectrometer (RS) and a hand-held energy dispersive X-ray fluorescence (EDXRF) analyzer. In addition, some black natural deposits with a dendritic pattern, typical of manganese compounds, were also in situ analysed with the aforementioned techniques to explore the possibility that post-Paleolithic people used minerals from the surroundings of the Los Chaparros rock-shelter to elaborate the paintings. The results obtained by the EDXRF analyses of black pigments showed differences in composition between a black Levantine deer, in which manganese was present as the main element, and a deep red Schematic pictograph that included manganese as the secondary element. The results of Principal Component Analysis (PCA) of collected EDXRF spectra showed similarities in the elemental composition between the manganese dendrite formations present in the rock-shelter and the black deer. In order to confirm this, the in situ analytical campaign was completed with some analysis in the laboratory by using micro-RS (μ-RS) and X-ray diffraction (XRD) on mineral samples having black crystallisations. Two specimens were taken, one from the black dendrite present in the same rock-shelter and the other from the Los Mases de Crivillén mining area (which is near to Los Chaparros). These analyses revealed that the characteristic bands of Mn–O and Mn–OH bending and stretching vibrations obtained in situ on the black pictograph were the same as those observed in the Raman spectra of the dendrite mineralization of Los Chaparros obtained in the laboratory by μ-RS.

Food safety monitoring and toxicological research of mycotoxins are still in need of large quantities of high-purity deoxynivalenol (DON). To attain this purpose, a rapid, economical and reproducible purification method was developed for large-scale production of DON from rice culture inoculated with a DON-producing Fusarium graminearum strain JYH. The inoculated rice culture was first extracted with acetonitrile–water (84/16, v/v). The extracts were evaporated to dryness on a rotary evaporator after ethyl acetate partitioning and then dissolved in water followed by the final purification procedure through preparative high performance liquid chromatography and montmorillonite treatment. A combined approach of ultraviolet spectrometry (UV), ultra high performance liquid chromatography (UHPLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy was applied for the multi-dimensional characterization of the target compound. As a result, the recovery of DON from the crude extract to the final product was up to 70%. An amount of 150 mg DON with the desirable purity of 98.93% could be obtained from 100 g of rice culture, which possessed identical immunochemical characteristics compared to a certified commercial DON standard. This proposed strategy might act as a valuable reference to obtain rather expensive compounds in a straightforward way.

Do voltammetry electrodes modified with MIPs really work? The role of large molecules: folic acid as a probe by Abigail Vasconcelos Pereira; Priscila Cervini; Éder Tadeu Gomes Cavalheiro (6658-6667).
There are many uncertainties concerning the use of molecularly imprinted polymers (MIPs) as electrode modifiers. In order to contribute to clarification of this issue, a large target molecule was used to prepare an MIP-modified composite electrode. The MIP was synthesized using folic acid (FA) as a template, and employed in the modification of composite electrodes based on graphite and polyurethane (GPU), varying its percentage from 2.5 to 10%. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to compare the performance of modified and unmodified electrodes. After optimization of the electrode composition, pH, and the electrolytic medium, the best results for which were obtained using 2.5% MIP, pH 4.5 and acetate buffer, respectively, differential pulse voltammograms were used to obtain analytical curves for electrodes modified with the 2.5% MIP or 2.5% non-imprinted polymer (NIP), as well as for the unmodified GPU electrode. Although the sensitivities were similar in all cases, the electrode modified with 2.5% MIP presented a lower limit of detection (LOD) of 0.17 μmol L−1 and 0.034 μmol L−1 for the cathodic peaks in −0.52 and −0.58 V (vs. SCE), respectively, under specific conditions, allowing the determination of FA in commercial pharmaceutical formulations with results that were in agreement with the official HPLC method. Finally, evaluation was made of the interference of a structurally similar molecule, the methotrexate, as well as small molecules containing functional groups similar to those present in the structure of FA, such as uric acid, ascorbic acid and dopamine. The 2.5% MIP electrode was more selective than the 2.5% NIP electrode.

Trace analysis of pesticides and an assessment of their occurrence in surface and drinking waters from the State of São Paulo (Brazil) by Cassiana C. Montagner; Cristiane Vidal; Raphael D. Acayaba; Wilson F. Jardim; Isabel C. S. F. Jardim; Gisela A. Umbuzeiro (6668-6677).
An efficient method based on solid phase extraction (SPE) and determination by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for simultaneous determination of 12 pesticides at trace levels in surface and drinking waters from the State of São Paulo (Brazil), which are likely to be contaminated due to the widespread use of these products. Several parameters that affect SPE and the analysis were studied, such as conditioning and elution solvents, sample pH, breakthrough volume and matrix effects. Method development was validated by several figures of merit. Recoveries from synthetic samples spiked at 150 ng L−1 and 1000 ng L−1 levels with difenoconazole, epoxiconazole, tebuconazole, atrazine, azoxystrobin, pyraclostrobin, picoxystrobin, trifloxystrobin, profenofos and fipronil varied from 73 to 99%, with intraday precision in the 5–24% range. A lower fortification level (10 ng L−1), close to detection limits, led to recoveries from 86–155%, which was considered acceptable for the purpose of trace analysis of environmental samples. Low detections limits (1–50 ng L−1) and quantification limits (2–180 ng L−1) were obtained. The method was applied for the determination of pesticide residues at the nanogram per liter level in samples of drinking water from 9 cities and in surface waters from 13 rivers of the State of São Paulo, Brazil. The results showed that the investigated waters are highly impacted with carbendazim and atrazine, which were the most frequently determined compounds.

Polycyclic aromatic hydrocarbons (PAHs) are contaminants frequently found in environmental waters. Owing to their toxic effects, it is important to develop analytical methodologies that allow their fast and reliable determination. Dispersive liquid–liquid microextraction (DLLME) allows a quick extraction of analytes with little organic solvent consumption. The best results regarding sample preparation are obtained when 40 μL of perchloroethylene is used as the extractant solvent and 1 mL methanol is used as the dispersive solvent; the extraction occurs one minute. Two-dimensional gas chromatography allows good separation of most PAH isomers. The methodology is characterized by good precision (<20% for the most of analytes) and recoveries (73–133%), while the LOD values (single ng L−1 for lighter PAHs) rapidly increase with increasing molar mass of the analytes. The methodology greenness assessment with NEMI and Ecoscale have shown that it may be considered as a green methodology. Real sample analysis proved that the procedure is convenient for PAHs determination in environmental and drinking water.

Reversed-phase high performance liquid chromatography as an alternative to animal bioassay for human thyrotropin potency determination by B. E. Almeida; R. Damiani; J. E. Oliveira; S. L. Dalmora; P. A. Torjesen; P. Bartolini; M. T. C. P. Ribela (6688-6694).
Reversed-phase high performance liquid chromatography (RP-HPLC) was compared to the in vivo bioassay (BA) based on TSH-induced T4 determination in mice. A linear relationship (BAμg = 0.9790 RP-HPLCμg− 0.052) with a highly significant correlation (r = 0.87; p < 0.001; n = 14 preparations) was found between these two methods. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for 11 other hTSH preparations was −2.01%, with a 95% confidence interval of −13.13% to +9.11%. This analysis included 5 totally or partially-degraded samples, indicating a useful correlation between the two determinations and, as expected, a higher sensitivity of the physical–chemical method. Interestingly, a commercial hTSH lot did not show any alteration 2 years post expiration date. These results demonstrate that RP-HPLC is a novel and viable alternative to the use of in vivo bioassays for hTSH potency determination, thus avoiding or reducing animal use.

Vortex-assisted extraction in tandem with dispersive liquid–liquid microextraction followed by GC-MS for determination of Achillea wilhelmsii essential oil by Hassan Sereshti; Abdollah Ghiasi; Mahdi Naderloo; Massoud Taghizadeh; Shakiba Darvish Alipoar Astaneh (6695-6701).
A simple, fast and efficient combined extraction/preconcentration method composed of vortex-assisted extraction (VAE) and dispersive liquid–liquid microextraction (DLLME) coupled with gas chromatography-mass spectrometry (GC-MS) was developed for determination of essential oil constituents of the plant Achillea wilhelmsii. Methanol and chloroform were used as the extraction and preconcentration solvents, respectively. The effective parameters of the extraction process were optimized by the one-at-a-time method. The optimal conditions were determined as 25 μL of chloroform, 7.5% (w/v) of salt concentration, and 2 min of extraction time. The linear dynamic range (LDR) for the oil components was within 0.01–100 and 0.1–100 mg L−1 characterized by determination coefficients (R2) between 0.9990 and 0.9998. The limits of detection (LODs) were from 3.0 to 30.0 μg L−1. The relative standard deviations (RSDs, n = 5) were 2.3–4.7%. The enhancement factors (EFs) ranged from 80 to 91. The extraction yield of the essential oil by hydrodistillation was 1.02% (w/w). The main components of the Achillea wilhelmsii essential oil were determined as: borneol (21.23%), cis-chrysanthenyl acetate (20.92%), camphor (9.24%), trans-chrysanthenyl acetate (8.63%) and 1,8-cineole (7.16%). The antimicrobial activity of the essential oil against 10 different species of bacteria with two most commonly used methods of the disc diffusion method (DDM) and the broth dilution method (BDM) was investigated. The essential oil showed inhibitory activity against Escherichia coli, Bacillus cereus, and Staphylococcus aureus with the highest activity for Escherichia coli.

This study describes the development of liquid chromatographic methods for the simultaneous separation of some of the most popular local anesthetics in pharmaceutical preparations and medical praxis (benzocaine, bupivacaine, chloroprocaine lidocaine, oxybuprocaine, prilocaine, procaine, propipocaine and tetracaine) based on a systematic approach using experimental design methodology in which one or more factors are changed at the same time. The strategy employs a chromatography modeling software for the simultaneous optimization of critical chromatographic parameters, which are gradient time tG, temperature T and the ternary composition of the organic eluent B. Three different stationary phases were investigated, which are: Kromasil C18, Prontosil C18-AQ and Luna Phenyl-Hexyl. To build the design space for each column, 12 initial experiments were carried out by systematical variation of the selected critical parameters simultaneously. The chromatographic conditions of these initial runs are based on two different gradient times (tG = 20 and 60 min, linear gradient system from 10–90% organic modifier B) each at two different temperatures (T = 25 and 40 °C) repeated at three different ternary composition of the eluent B (a) 100% acetonitrile, (b) acetonitrile–methanol (50 : 50) (v/v), and (c) 100% methanol. In all experiments the pH value of the eluent A (20 mM phosphate buffer) was kept at 3.0. The mixture of local anesthetics could be well separated on all three stationary phases. Although not demonstrated in this paper, this method should be suitable for the analysis of LAs in pharmaceutical preparations or to detect them in some illegal cosmetics. The results showed that the selectivity and the elution order were similar on Kromasil C18 and Prontosil C18-AQ. On the other hand, a unique selectivity is resulted on Luna Phenyl-Hexyl, which shows, depending on the analytes, some additional interactions, since the separation mechanism on this column is influenced by its different steric and polar properties compared to the separation mechanism of alkyl-bonded phases. The predictions and real experiments were strongly correlated with an average absolute error (ΔtR) of 0.13 min (<8 s).

Mesoporous silica particle embedded functional graphene oxide as an efficient platform for urea biosensing by Shiju Abraham; Valerian Ciobota; Saurabh Srivastava; Sunil K. Srivastava; Rajesh K. Singh; Jan Dellith; B. D. Malhotra; Michael Schmitt; Jürgen Popp; Anchal Srivastava (6711-6720).
The mesoporous silica particle embedded graphene oxide (GO) hybrid system is a promising platform for electrochemical biosensing owing to its large 2-dimensional structure, fast electron transfer kinetics, improved hydrophilic nature and surplus functional groups. Here, GO sheets were synthesized by Hummer's improved method and sub-micron sized homogeneous mesoporous silica (SiO2) particles were prepared by Stober's method. The SiO2 particles were embedded on the GO surfaces and were optimized with different concentrations for better applicability and hydrophilicity. Micro-structural and spectroscopic characterization of as-synthesized materials was carried out to confirm the successful synthesis as well as the functionalities required for biosensing. Scanning electron microscopy investigations suggest that the average size of the SiO2 particles decorated on the GO surface is ∼500 nm. Raman investigation provides information regarding the increase in defects and disorder on the GO surface with the increase in the SiO2 content. The optimized GO–SiO2 (GOS) composite electrode was prepared by the electrophoretic deposition technique and was used for the attachment of urease and glutamate dehydrogenase enzymes for urea detection employing the cyclic voltammetry method. The reproducibility, specificity and stability of the fabricated biosensor were found to be excellent for the urea sensing. Such an easy and cost effective material based GOS urea sensor showed a high sensitivity (2.6 μA mM−1 cm−2) and a good detection limit (14 mg dL−1).

Circular dichroism (CD) spectroscopy is a quick method for measuring data that can be used to determine the average secondary structures of proteins, probe their interactions with their environment, and aid in drug discovery. This paper describes the operation and testing of a self-organising map (SOM) structure-fitting methodology named Secondary Structure Neural Network (SSNN), which is a methodology for estimating protein secondary structure from CD spectra of unknown proteins using CD spectra of proteins with known X-ray structures. SSNN comes in two standalone MATLAB applications for estimating unknown proteins' structures, one that uses a pre-trained map and one that begins by training the SOM with a reference set of the user's choice. These are available at http://www2.warwick.ac.uk/fac/sci/chemistry/research/arodger/arodgergroup/research_intro/instrumentation/ssnn/ as SSNNGUI and SSNN1_2 respectively. They are available for both Macintosh and Windows formats with two reference sets: one obtained from the CDPro website, referred to as CDDATA.48 which has 48 protein spectra and structures, and one with 53 proteins (CDDATA.48 with 5 additional spectra). Here we compare SSNN with CDSSTR, a widely-used secondary structure methodology, and describe how to use the standalone SSNN applications. Current input format is Δε per amino acid residue from 240 nm to 190 nm in 1 nm steps for the known and unknown proteins and a vector summarising the secondary structure elements of the known proteins. The format is readily modified to include input data with e.g. extended wavelength ranges or different assignment of secondary structures.

A simple, stability-indicating, reversed phase liquid chromatographic method has been developed for the determination of fluphenazine hydrochloride in the presence of its oxidative and ultraviolet degradation products. Reversed phase chromatography was conducted using an ODS C18 (150 × 4.6 mm id) column at ambient temperature with UV-detection at 250 nm. A mobile phase consisting of 0.03 M potassium dihydrogen phosphate buffer–acetonitrile (40 : 60, v/v) adjusted to pH 4 was used for the separation of the studied drug and its degradation products at a flow rate of 1 mL min−1. The method showed a good linearity over the concentration range of 2.0–20.0 μg mL−1 with a detection limit (LOD) of 0.8 μg mL−1 and a quantification limit (LOQ) of 1.5 μg mL−1. The proposed method was successfully validated and applied for the analysis of the drug in its commercial dosage forms; the obtained results were favorably compared with those obtained by the official and comparison methods. Moreover, the method was utilized to investigate the kinetics of the oxidative degradation of the drug. The first-order rate constant, half-life time, and activation energy of the degradation were calculated.

A novel trimethylstearylammonium bromide (TSAB) coated Fe3O4/SiO2 magnetic nanocomposite was fabricated and used as the adsorbent for the magnetic solid-phase extraction of adriamycin hydrochloride (ADR) in human plasma and urine with high performance liquid chromatography-fluorescence detection (HPLC-FLD). The factors influencing the extraction efficiency were examined including the pH value, extraction time, elution solvent, etc. A good linearity was presented by HPLC-FLD in the range of 0.5–10.0 μg mL−1 for ADR, with the correlation coefficient of 0.998 (R2). The relative standard deviation was 4.27%. The limit of detection and limit of quantitation were 5.05 and 16.82 ng mL−1, respectively. The feasibility of the developed method was further validated by extraction of ADR in plasma and urine samples. The recoveries were in the range of 76.5–94.0% and 77.9–96.0% for plasma and urine samples, respectively.

A liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for determination of tulathromycin in swine tissues has been developed. Prior to the instrumental analysis, the sample preparation technique involved extraction with a solution of meta-phosphoric acid followed by solid-phase extraction clean-up using a polymeric mixed-mode strong cation-exchange sorbent. The chromatographic separation was performed on a C8 chromatographic column with a gradient elution mode. Mass spectral acquisition was performed in selective multiple reaction monitoring mode by using a triple quadrupole mass spectrometer. The method has been characterized by providing the parameters of precision, accuracy, specificity, decision limit (CCα) and detection capability (CCβ) according to the Commission Decision 2002/657/CE. The calibration curves were linear in the range of 10–9000 μg kg−1. The recoveries ranged from 92.9% to 102.1%. The repeatability was below 8.0% and the within-laboratory reproducibility was lower than 9.2%. The limits of detection ranged from 2 to 4 μg kg−1 and the limit of quantification was 10 μg kg−1. The presented analytical method can be used for measuring the tulathromycin depletion in swine tissues (kidney, liver and muscle) over time.

Thorough analysis of Iranian spearmint essential oil: combination of chemometrics and gas chromatography-mass spectrometry by Mehdi Jalali-Heravi; Roudabeh Sadat Moazeni-Pourasil; Hassan Sereshti (6753-6759).
The components of Iranian spearmint essential oil were identified and quantified using gas chromatography-mass spectrometry (GC-MS) combined with chemometric tools. A multivariate curve resolution (MCR) approach was used to overcome the problems of background, baseline offset and overlapping/embedded peaks in GC-MS. The peaks were resolved using heuristic evolving latent projection (HELP) and multivariate curve resolution-alternating least squares (MCR-ALS) techniques. A new pure variable selection method named multivariate curve resolution-objective function equal to zero (MCR-FEQZ) is introduced and was applied in this work. The performance of this technique was evaluated and confirmed using MCR-ALS. The analysis of GC-MS data with the help of MCR techniques revealed that the five most abundant constituents of the Iranian spearmint essential oil are (+)-carvone (73.20%), limonen (14.63%), β-bourbonene (1.13%), cineole (1.10%) and 3-terpinolenone (0.82%). The results show that this essential oil could be a great source of carvone and limonene.

The origin of progesterone in butter was investigated using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). GC/C/IRMS has been rarely used to investigate the origin of natural steroid hormones, which are often found in some foods of animal origin used as feed additives. In this study, an effective pretreatment method for a butter sample was developed using gel permeation chromatography (GPC). The butter sample was dissolved with ethyl acetate–cyclohexane (1/1, v/v). After GPC pretreatment and HPLC purification, the sample was analyzed for progesterone on a GC/C/IRMS system. The δ13C values of endogenous progesterone in butter were found in the range from −26.77 ± 0.26‰ to −24.78 ± 0.32‰ and the δ13C value of exogenous progesterone spiked in the sample was about −30.57 ± 0.12‰. One-way analysis of variance (ANOVA, P-value = 0.01 < 0.05) indicated that there was a highly significant difference between the endogenous and exogenous δ13C values of progesterone. The sensitivity of the method for progesterone was determined to be from 10 to 100 ng. The within-batch precision of the method ranged from 0.09 to 0.15‰ and the between-batch precision was 0.11‰. We found that the HPLC fraction collection step was a critical factor affecting the isotopic fractionation and measurement accuracy. GC/C/IRMS can be a powerful tool to investigate the origin of natural hormones.

Highly efficient turn-on fluorescence detection of zinc(ii) based on multi-ligand metal chelation by Jian Sun; Tao Yu; Huan Yu; Mingtai Sun; Huihui Li; Zhongping Zhang; Hui Jiang; Suhua Wang (6768-6773).
We report that the simple Schiff base molecule, 2-(((pyridin-2-ylmethyl)imino)methyl)phenol, can efficiently chelate zinc(ii) to produce a highly fluorescent complex, ML2, which could be useful in the development of turn-on fluorescence detection of zinc(ii). The compound initially shows very weak fluorescence, however, the coordination with the zinc ion inhibits the CN bond isomerization and subsequently enhances the fluorescence efficiency. Based on the turn-on fluorescence, the limit of detection for zinc(ii) was measured to be 62 nM, which is lower than the allowable level of zinc (∼70 μM) in drinking water set by the U.S. Environmental Protection Agency. We tried to make fluorescence test strips by immobilizing the compound in silica gel plates and demonstrated the application for visualization of zinc screening in water and aerosol samples. The visual limit of detection was estimated to be as low as 9 μM.

Electrochemical determination of selected antihypertensive and antituberculosis drugs at a tyrosine-modified electrode by Srikanth Cheemalapati; Balamurugan Devadas; Shen-Ming Chen; M. Ajmal Ali; Fahad M. A. Al-Hemaid (6774-6782).
A simple and sensitive electrochemical method was proposed for the determination of hydralazine hydrochloride (HDH), isoniazid (ISN), ethambutol hydrochloride (EBH) and pyrazinamide (PZM). For the first time, a tyrosine (Tyr)-modified glassy carbon electrode (GCE) was employed for the determination of HDH, ISN, PZM and EBH by the differential pulse voltammetric (DPV) technique. The proposed modified electrode showed strong electrocatalytic activity towards the above-mentioned drugs with a higher peak enhancement than that of an unmodified electrode. The practicality of the proposed electrode for the detection of HDH, ISN, PZM and EBH in human urine and blood serum samples was successfully demonstrated using the DPV technique. The applicability of the proposed method was verified with commercially available pharmaceutical tablets and the obtained results were in good agreement with the claimed label amounts of the tablets. From these results, it is clearly evident that the proposed electrode shows good catalytic activity towards HDH, ISN, PZM and EBH. In addition, this method could be used in the future for the accurate detection of HDH, ISN, PZM, and EBH in clinical and pharmaceutical industries.

Ionic liquid (IL)-coated magnetic Fe3O4 nanoparticles (NPs) as an adsorbent of mixed hemimicelles solid-phase extraction (SPE) are proposed for the preconcentration of five organic contaminants (catechol, bisphenol A, 2,4-dichlorophenol, 1,3,5-trichlorobenzene and acrylonitrile) from environmental water sample prior to high-performance liquid chromatography (HPLC). In the present study, mixed hemimicelles formed by IL-coated Fe3O4 NPs showed a great adsorptive tendency towards the target analytes, and then the analyte-adsorbed mixed hemimicelles could be readily isolated from the sample solution by a magnet. Note that the proposed method could greatly simplify the operation and reduce the whole pretreatment time. Moreover, several parameters affecting extraction efficiency, such as the amount of Fe3O4 and [C8MIm]Br IL, the type and pH of desorption solvent, and the extraction time and salt effect, were investigated. Under optimum conditions, satisfactory recoveries for the target analytes were in the range of 90%–102% with 3.3%–8.1% relative standard deviations (RSD, n = 6), 90%–111% with 2.1%–7.6% RSD, 92%–99% with 5.5%–13.5% RSD at 0.01 μg mL−1, 0.1 μg mL−1 and 100.0 μg mL−1 levels, respectively. The limits of detection (LODs) for the target analytes were in the range of 0.01–0.07 μg L−1.

Scissor-based fluorescent detection of pepsin using lysozyme-stabilized Au nanoclusters by Wei Li; Zhao Gao; Rongxin Su; Wei Qi; Libing Wang; Zhimin He (6789-6795).
Pepsin is an active digestive enzyme present in the acidic environment of animal stomachs, and has been widely used to prepare bioactive peptides in the food industry. In this work, a simple fluorescence sensor for scissor-based detection of pepsin activity was developed by using lysozyme-stabilized gold nanoclusters (AuNCs@Lyz) in aqueous media. Under acidic conditions (pH 3.0), enzymatic digestion of AuNCs@Lyz with pepsin results in a significant decrease of fluorescence intensity. Notably, its acidic environment not only helps maintain the maximum fluorescence of gold nanoclusters, but also ensures the highest enzymatic activity of pepsin. In addition to offering high selectivity because of the unique proteolytic action of pepsin under acidic conditions, this facile method provides high sensitivity. With the sensing system, the linear range for pepsin detection is found to be 1 μg mL−1 to 100 μg mL−1, with a detection limit of 0.256 μg mL−1 at a signal-to-noise ratio of 3. Furthermore, the AuNCs@Lyz based fluorescent sensing system could find applications in highly sensitive and selective detection of pepsin in food and biological samples.

Label-free and sensitive electrochemiluminescence aptasensor for the determination of 17β-estradiol based on a competitive assay with cDNA amplification by Jing-Jing Zhang; Jun-Tao Cao; Gui-Fang Shi; Ke-Jing Huang; Yan-Ming Liu; Yong-Hong Chen (6796-6801).
A label-free competitive electrochemiluminescence (ECL) aptasensor for the determination of 17β-estradiol (E2) was developed based on the immobilization of thiol-capped E2 aptamers onto a gold electrode. A complementary DNA (cDNA) was designed to be used as a detection probe to bind with any unbound E2 aptamer. Tris(2,2′-bipyridyl)ruthenium(ii) (Ru(bpy)32+) was used as an ECL indicator and was electrostatically bound to the E2 aptamer; it competes with the cDNA for aptamer binding sites. With less E2, increased amounts of cDNA can bind to the E2 aptamer and adsorb more Ru(bpy)32+, greatly enhancing the ECL intensity. With more E2, the aptamer will capture E2 and bind less cDNA with an accompanying decrease in Ru(bpy)32+ and a decrease in ECL intensity. Thus, a competitive aptasensor based on the specific binding of the E2 aptamer to E2 and cDNA was achieved. The biosensor for E2 possesses a widely linear detection range from 0.01 to 10 nmol L−1 with a detection limit of 1.1 × 10−12 mol L−1 (S/N = 3). The application of the present protocol was demonstrated by determining E2 in human serum, human urine, and tap water samples with recoveries of 89.8–100.0%, 90.0–103.5% and 89.5–95.0%, respectively. The proposed method provides a powerful tool for the rapid and sensitive detection of small molecules in biological and environmental samples.

Sensitive quantification and visual detection of bacteria using CdSe/ZnS@SiO2 nanoparticles as fluorescent probes by Renjie Wang; Yi Xu; Yan Jiang; Na Chuan; Xi Su; Jingou Ji (6802-6808).
CdSe/ZnS@SiO2–NH2 composite nanoparticles (FNPs) were proposed and used as fluorescent markers for bacteria detection. Salmonella typhimurium acted as representative sample in this paper. It was shown that hydrophobic CdSe/ZnS quantum dots (QDs) were incorporated into SiO2 spheres by using a self-modified reverse-microemulsion technique. FNPs could be successfully covalently conjugated with the bacteria by glutaraldehyde in a two-step strategy. A good linear relationship between the concentration of Salmonella typhimurium and fluorescence intensity was obtained in the range of 6.6 × 102 to 6.6 × 104 cfu mL−1, and the equation was I = 0.1331 log C− 0.2017 with R2 = 0.9974. The detection limit was 3.3 × 102 cfu mL−1, and this method could be applied to other bacteria detection as well. In order to further reduce the detection limit and achieve better visual determination performance, an integrated dielectrophoresis (DEP) microfluidic chip and relative microsystem was established. The FNP-labeled bacteria could be enriched along the edges of interdigitated microelectrodes in the micro-channel by positive DEP and could be counted under the fluorescence microscope.

A fluorescent aptasensor for sensitive detection of human hepatocellular carcinoma SMMC-7721 cells based on graphene oxide by Qin Xie; Yuyu Tan; Qiuping Guo; Kemin Wang; Baoyin Yuan; Jun Wan; Xiayu Zhao (6809-6814).
In this work, we reported a simple, rapid and sensitive fluorescence method for the direct detection of cancer cells using a graphene oxide (GO)-based aptasensor. This method took advantage of the exceptional quenching capability of GO for dye-labelled aptamers, and an aptamer was employed as the molecular recognition element. As proof of concept, an aptamer sequence was selected using a cell-based SELEX strategy in our laboratory for human liver cancer cell line SMMC-7721 with high specificity and high affinity, which was applied in this method. Samples with the target cells show high fluorescence intensity, while non-target samples still showed low fluorescence intensity. This GO-based aptasensor exhibited high sensitivity and specificity with a detection limit of 200 SMMC-7721 cells in 200 μL binding buffer. In addition, our strategy can directly detect target cells without washing and separation. Furthermore, the future design of detectors for other cancer cells only requires changing the recognition element for the target cell. More importantly, flow cytometry was used for the specific recognition of target cells from a mixture of cells in fetal bovine serum to demonstrate the potential application of this method for medical diagnostics.

This study first describes the development of a rapid and accurate high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the quantification of astragaloside II (AST II) in rat plasma. The assay involved a simple protein precipitation (PPT) step with methanol–acetonitrile (50 : 50, v/v) and a gradient elution using a mobile phase consisting of water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. Chromatographic separation was successfully achieved on an Agilent Zorbax XDB C18 column (2.1 mm × 50 mm, 3.5 μm) with a flow rate of 0.50 mL min−1. Multiple reaction monitoring (MRM) was based on the transitions of m/z = 827.3 → 143.2 for AST II and 386.3 → 122.3 for buspirone (IS). The assay was validated to demonstrate the specificity, linearity, recovery, accuracy, precision and stability. The lower limit of quantification (LLOQ) was 5.0 ng mL−1 in 50 μL of rat plasma. The developed and validated method has been successfully applied to the quantification and pharmacokinetic study of AST II in rats after intravenous and oral administration of AST II. The oral absolute bioavailability (F) of AST II was calculated to be 0.79 ± 0.16% with an elimination half-life (t1/2) value of 1.92 ± 0.30 h, suggesting its poor absorption and/or strong metabolism in vivo.

CoMFA and CoMSIA studies on a series of fluroquinolone derivatives for potential anti-inflammatory activity by Asia Naz; Hina Shamshad; Zaheer ul Haq; Farhan Ahmed Siddiqui (6823-6831).
Three-dimensional quantitative structure–activity relationships using CoMFA and CoMSIA were developed for a series of 28 fluroquinolone derivatives for prediction of anti-inflammatory activity. QSAR models with high squared correlation coefficients of up to 0.962 for CoMFA, 0.989 for CoMSIA-I and 0.987 for CoMSIA-II were established. The robustness of the model was confirmed with the help of leave-one-out cross-validation having values of 0.554 for CoMFA, 0.6 for CoMSIA-I and 0.597 for CoMSIA-II, respectively. Theoretical results were in accordance with the experimental data. Developed models highlighted the importance of steric, electrostatic, hydrophobic and donor descriptors for anti-inflammatory activity.

Classification of multi-family enzymes by multi-label machine learning and sequence-based descriptors by Yuelong Wang; Runyu Jing; Yongpan Hua; Yuanyuan Fu; Xu Dai; Liqiu Huang; Menglong Li (6832-6840).
Multi-family enzymes are of great importance in life, disease and other domains. However, in terms of the classification of enzymes, the information of multi-family enzymes is always removed from the dataset to account for the limitation of traditional single-label prediction methods. In order to predict multiple classes of multi-family enzymes, we adopted two multi-label learning algorithms, namely RAkEL-RF and MLKNN, and two types of protein descriptors, namely CTD and PseAAC, to generate four predictors, RAkEL-RF-CTD, RAkEL-RF-PseAAC, MLKNN-CTD and MLKNN-PseAAC. When the four predictors were tested on a training set with 10-fold cross validation, the overall success rates reached 97.99%, 96.07%, 96.01% and 95.31%, respectively. For the independent test set, the corresponding rates reached 97.57%, 95.03%, 95.9% and 93.9%, respectively. In conclusion, it proved the outstanding prediction capability and robustness of our predictors from the extremely small difference between two sets for each predictor and the relatively higher accuracy. In addition, three of seven pairs of homologous enzymes with different functions and eighteen of twenty-three distantly related enzymes with a similar family were correctly classified by the RAkEL-RF-CTD predictor. These results indicated the extensive applicability of our predictors.

This paper reports the use of chromatographic profiles of breath volatiles to determine disease markers in lung cancer patients and healthy volunteers. The volatile fraction was isolated by headspace solid-phase microextraction (HS-SPME) and analyzed by flow-modulated comprehensive two-dimensional gas chromatography and flame ionization (GC × GC-FID). Following the experiments, collected data were transformed, and partial least-squares discriminant analysis (PLS-DA) as well as Mann–Whitney Test were carried out to model the data and discover breath metabolites with a significant concentration difference between patients and healthy subjects. Using the abovementioned method, lung cancer patients and healthy controls could be correctly distinguished based on metabolic VOCs abnormality in human breath. Five potential target compounds including acetone, isoprene, methanol, pentane and propanol were identified. Lung cancer patients show higher concentrations of propanol (7415.3 ng L−1), acetone (1811.6 ng L−1) and methanol (225 ng L−1) compared with those of healthy volunteers (1975.3 ng L−1, 579.9 ng L−1, 76.8 ng L−1, respectively). In addition, there is no significant relationship between breath VOCs and gender or body mass index (BMI). This approach will facilitate the comparison of complex breath VOC profiles and diseases. These findings may offer valuable and reliable information for the early diagnosis and prognosis of lung cancer.

Microctis Folium (MF), the leaves of Microcos paniculata L., which is widely used as a traditional Chinese medicine and a healthy tea, show various biological activities including antioxidant, cardiovascular risk, and so on. However, few studies on the quality control for this herbal medicine have been reported. In this paper, a reliable and sensitive online high-performance liquid chromatography coupled with diode array detection and electrospray ionization tandem ion trap mass spectrometry (HPLC-DAD-ESI-MS/MS) method for qualitative and quantitative evaluation of the characteristic chemical constituents of MF was developed. 32 compounds including 10 flavans, 16 flavonoid glycosides, 5 lignan glycosides and 1 megastigmadien glycoside were identified from MF, among which lignan glycosides and megastigmadien glycoside were first reported from the genus Microcos. Besides, based on HPLC-DAD-ESI-MS/MS data, a standardized HPLC fingerprint and a quantitative method were established by analyzing 13 batches of commercial herbal samples by HPLC with ultraviolet-visible (UV) detection method. Meanwhile, three major bioactive constituents including kaempferol-3-O-β-d-glucoside, narcissi and isorhamnetin-3-O-β-d-glucoside in MF were simultaneously determined with the same method, which could differentiate good quality batches from counterfeits. The results revealed that this HPLC-DAD-ESI-MS/MS analytical method established could be adopted as the comprehensive quality control method of MF.

Adulteration of high-price edible oils has become a focus of attention and a tough problem in food trade and consumption all over the world. Therefore, there is a great demand for detecting oil adulteration to protect interests and rights of customers and safeguard their health. In this study, free phytosterol profiles of peanut, soybean, rapeseed, and sunflower seed oils were established by SPE-multidimensional gas chromatography coupled with time-of-flight mass spectrometry (GC-GC-TOF/MS) and employed to classify the four edible oils with the help of unsupervised (principal component analysis and hierarchical clustering analysis) and supervised (random forests) multivariate statistical methods. The results indicated that free phytosterol profiles of edible oils could help classify the four edible oils into four groups completely, and therefore, could be used as important markers of the oils studied. Moreover, a simulated data test revealed that free phytosterol profiles could also be used to detect adulteration of peanut oil with 5% soybean oil, which was simulated by the Monte Carlo method.

A new method for the analysis of niclosamide (NIC) and its primary degradates 2-chloro-4-nitroaniline (2C4NA), aminoniclosamide (AN), hydroxyniclosamide (HN) and 5-chlorosalicylic acid (5CSA) in water was developed using direct injection LC-MS/MS. Methanol and acetonitrile mobile phases were compared. Methanol was superior for both separation and sensitivity for all chemicals. LLoQs for all chemicals were 3–50 times better in methanol than in acetonitrile, and baseline separation was observed for HN and 5CSA in methanol but not acetonitrile. The LLoQ for NIC in the current study was approximately 20 times lower than that previously reported using LC-MS/MS methodology, and 10–250 times lower for all chemicals than obtained by HPLC-UV/visible detection. The method reported in the current study relies upon smaller injection volumes and direct injection of water, eliminating time consuming clean-up steps and increasing sample throughput. The current method is also more selective for all four chemicals than existing HPLC-UV/visible methods, and is not susceptible to spectral interferences from DOM. The use of a shorter LC column with core shell particles resulted in shorter run times and lower mobile phase consumption than previously reported methods for NIC, which rely on traditional porous particle columns.

A fingerprint analysis method was developed for the quality control of Salvia yunnanensis roots by HPLC. The HPLC separation was performed on a Hypersil ODS (4.6 mm × 250 mm, 5 μm) column by gradient elution with acetonitrile–water as the mobile phase, at the temperature of 30 °C and a flow rate of 1.0 mL min−1. A satisfactory separation of 21 common peaks was achieved within 60 min. Similarity evaluation was performed by professional software named Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine, and the similarity among 11 batches of samples was no less than 0.92. The results of similarity evaluation confirmed that the quality of Salvia yunnanensis roots was stable, and the developed fingerprint analysis was precise and efficient for the quality control of Salvia yunnanensis roots.

A glassy carbon electrode was modified with nickel-ferrite magnetic nanoparticles and decorated with multiwall carbon nanotubes (NiFe2O4–MWCNTs). Differential pulse voltammetry was then used to investigate the electrochemical behavior of epinephrine at the surface of the modified electrode. The properties of the nanocomposite were also characterized using different techniques. The electrode showed an excellent synergic effect on epinephrine oxidation. At the optimum pH level, the electrode's response in 0.1 mol L−1 phosphate solution was proportional to the concentration of epinephrine in the range of 0.9–800.0 μmol L−1 with a detection limit of 0.09 μmol L−1. The effects of different potentially interfering substances on the epinephrine signal were also studied. Finally, the sensor was evaluated with respect to its reproducibility and stability. It was found that the modified electrode had a good sensitivity, selectivity, and reproducibility for the determination of epinephrine in real samples.

Due to the significance of bleomycins (BLMs) in the treatment of a variety of cancers, it is highly desirable to develop a sensitive method to quantitatively determine the content of BLMs in both pharmaceutical analysis and clinical samples. Here, a simple, rapid, and convenient electrochemical DNA sensor for determination of BLMs was developed based on 4-mercaptophenyl boronic acid-capped gold nanoparticles (MBA-AuNPs) and dopamine-capped gold nanoparticles (DA-AuNPs) as a dual-amplification element. In the presence of Fe(ii)·BLMs, the electrochemical DNA sensor undergoes an irreversible cleavage event, which can result in a significant decrease of the oxidation peak current of dopamine. This proposed sensor reveals a wide linear range from 0.07 nM to 910 nM (R = 0.9958) and a low detectable limit (0.02 nM) for the determination of BLMs. It also exhibits a good performance in serum samples. Based on the high sensitivity and specificity of this sensor, and the inherent characteristics (cost-effective and simple-to-implement) of the electrochemical technique, the developed DNA sensor shows distinct advantages over conventional methods and is a promising method for the determination of trace amounts of BLMs in both pharmaceutical analysis and clinical samples.

A thermal desorption method followed by GC/MS analysis has been developed for efficient analysis of levoglucosan and PAHs in particulate matter. The method requires sample derivatization using N,O-bis(trimethylsilyl)trifluoroacetamide with 1% of trimethylchlorosilane. The reaction is carried out directly in the liner of a commercial thermo-desorption system on 2.1 mm PM2.5 filter cuts under optimized conditions of temperature, 20 °C, and reaction time, 5 minutes. A low temperature of 150 °C is used for desorption, minimizing possible artefact formation and allowing simultaneous detection of PAHs. The method is evaluated on a standard reference material NIST 1649a and applied on ambient air samples. For a sampled air volume of circa 200 L, the overall analytical expanded uncertainty (OEU) for levoglucosan at a concentration of 500 ng m−3 is 25%, while its detection limit (LOD) is circa 50 ng m−3. In the case of benzo[a]pyrene, OEU at 1 ng m−3 is 20% and its LOD 95 pg m−3.

Development of green miniaturized dispersive ionic liquid nano-emulsion method for preconcentration of cadmium from canal and waste water samples prior to coupling with graphite furnace atomic absorption spectrometry by Naeemullah; Tasneem Gul Kazi; Hassan Imran Afridi; Faheem Shah; Sadaf Sadia Arain; Abdul Haleem Panhwar; Salma Aslam Arain; Mohammad Balal Arain (6909-6915).
A green miniaturized dispersive ionic liquid nano-emulsion extraction (NDILE) method has been developed for the preconcentration of trace levels of cadmium (Cd) in canal, tap and waste water samples. In the proposed approach, 1-butyl-3-methylimidazolium hexafluorophosphate, [C4mim][PF6], was dispersed into nano-emulsions by a low-energy emulsification phase inversion method, stabilized by a nonionic surfactant (TX-114). The influence of variables such as ionic liquid volume, concentration of the surfactant, stirring rate and time was studied and optimized. The morphology of the nano-emulsion system and its stability were evaluated through visual assessment and optical light microscopy. Ionic liquid nano-emulsion (ILN) was used as a dispersive and extractive medium, due to its small size and high surface area, to enhance the extraction efficiency of Cd complexed with 8-hydroxyquinoline from aqueous media. Separation of stable ILN could be induced by the micellar cloud point extraction approach. Under optimum conditions, the limit of detection and enhancement factor were found to be 0.48 ng L−1 and 180, respectively. Validation of the developed method was carried out by Cd determination using a certified reference material (SLRS-4 Riverine water) and the results were found to be in good agreement with the certified values. The proposed novel method was successfully applied for determination of Cd in real water samples.

In this work we report an easy and simple green synthesis method for gold nanoparticles (GNPs) using willow tree bark extract. Willow tree bark includes aspirin, which acts as a reducing agent. In the present method, there is no need to add any stabilizing agent such as a surfactant to stabilize the synthesized GNPs. Therefore, as an alternative to chemical synthesis, the green synthesis of GNPs with willow tree bark extract is beneficial for its potential biological and medical applications. The optimum conditions for GNP synthesis were obtained by studying the pH and the amount of willow tree bark extract solution. The characteristics and morphologies of GNPs were investigated by UV-Vis spectroscopy and transmission electron microscopy. The interaction between the synthesized GNPs and cysteine was introduced as a new and high-potential colourimetric sensor for the selective recognition and monitoring of cysteine, among other amino acids. The sensitivity and selectivity of GNPs toward cysteine in comparison to other amino acids were studied.

Based on the prepared group-specific antibodies against polychlorinated biphenyls (PCBs), a direct competitive real-time fluorescent quantitative immuno-polymerase chain reaction (rt-IPCR) assay was developed. The purpose of the assay was the determination of multi-residue PCBs in indoor air samples. In the assay, male New Zealand white rabbits were immunized with an immune antigen mixture composed of PCB12-O-BSA, PCB37-O-BSA, and PCB77-O-BSA. The specific polyclonal antibodies (pAbs) to multi-residue PCBs were obtained and used to develop a direct competitive rt-IPCR assay. The specificities of the pAbs were examined by the indirect competitive enzyme-linked immunosorbent assay (id-ELISA). The assays were found to be highly specific for PCB congeners as well as Aroclors 1248 and 1242. The effect of optimal reagent concentrations on reducing background fluorescence was also investigated. Using the optimized assay, a standard curve for Aroclor 1248 was prepared. The linear range for the determination of PCBs was 10 to 106 fg mL−1 with a correlation coefficient of 0.98 and a detection limit of 10.25 fg mL−1. The entire procedure was then evaluated using spiked air samples. The rt-IPCR results for the air samples were confirmed by gas chromatography/mass spectrometry and ELISA. Recovery was lower or higher with agitation but would still be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for detection of PCBs in air samples.

For the trace determination and automated continuous monitoring of volatile organic compounds (VOCs) in surface water, a large volume-direct aqueous injection-gas chromatography (LV-DAI-GC) method was optimized for the following parameters: packed sorbent, inlet temperature, injection volume, detector configurations and sample renewal. Using the packed sorbent lab-made with lithium chloride and diatomite 101 W, at an inlet temperature of 140 °C, 14 typical VOCs were separated from a 50 μL water matrix in 0.3 min. Coupled with a flame ionization detector (FID), an electron capture detector (ECD) and a flame photometric detector (FPD), the method quantification limits (MQLs) for the 14 typical VOCs were 0.008–7 μg L−1. For the samples from the simulated VOC pollution incidents, the relative standard deviations (RSDs) and relative errors (REs) were 5.9% and 9.5%, respectively. For real samples from a river polluted with VOCs, the quantitative results using LV-DAI-GC agreed well with those of purge-and-trap GC (PT-GC). The optimized reliable LV-DAI-GC could be applied to the automated continuous monitoring of VOCs in surface water and an early warning of VOC pollution incidents.

Rapid simultaneous determination of herbicides in human serum by UPLC-ESI-MS by Xinfeng Dong; Zhihong Shi; Shuxuan Liang; Hanwen Sun (6939-6947).
A simple, rapid and reliable method was developed for multi-class and multi-residue analysis of herbicides in human serum by ultra-performance, liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS). Serum sample preparation was carried out by one-step protein precipitation and analytes extraction using acetonitrile. After centrifugation, an aliquot of 5 μL of supernatant was injected into a C18 column for the separation of 22 kinds of triazine and phenylurea herbicides using gradient program with water–acetonitrile as the mobile phase and the separation of 29 kinds of herbicides using gradient program with 5 mM ammonium acetate aqueous solution containing 0.1% v/v formic acid–acetonitrile as the mobile phase. An excellent linearity of most herbicides was observed from 0.1 μg L−1 up to 10.0 μg L−1. The limits of detection (LODs) in serum ranged from 0.03 to 6.00 μg L−1, and the limits of quantification (LOQs) ranged from 0.10 to 18.0 μg L−1. Intra- and inter-day precisions at three spiked levels were satisfactory for the 51 herbicides with the RSD of 1.02–10.0% and 1.09–12.0%, respectively. Extraction recoveries of 51 herbicides were satisfactory and ranged from 63.6% to 109% at the three spiked levels with RSDs of 1.06% to 12.0%. This UPLC-ESI-MS method is simple, accurate, and useful for multi-class multi-residue determination of herbicides and benefits clinical analysis and diagnosis.

Fluorescence quenching studies of CTAB stabilized perylene nanoparticles for the determination of Cr(vi) from environmental samples: spectroscopic approach by Dattatray K. Dalavi; D. P. Bhopate; A. S. Bagawan; A. H. Gore; N. K. Desai; A. A. Kamble; P. G. Mahajan; G. B. Kolekar; Shivajirao R. Patil (6948-6955).
Cetyl trimethyl ammonium bromide (CTAB) stabilized perylene nanoparticles (PNPs) were prepared by a modified reprecipitation method in aqueous solution under ultrasonic treatment. A spectrofluorimetric method for the quantitative determination of hexavalent chromium (Cr(vi), dichromate species) based on the fluorescence (FL) quenching of CTAB-stabilized PNPs (CTAB-PNPs) in aqueous solution was proposed. Under the most favourable conditions, the FL intensity of PNPs monitored at an excitation wavelength of λex = 382 nm was quenched by the successive addition of increasing concentrations of dichromate ions. The FL quenching results were found to fit the Stern–Volmer (S–V) relationship in the range of 0.5–50 μg mL−1 with a correlation coefficient of 0.9997. The limit of detection (LOD) was 0.008 μg mL−1. The method based on FL quenching was successfully applied for the quantitative analysis of Cr(vi) in water samples collected from different environments.

A sensitive, wide-scope, and trace determination of nine residual pharmaceuticals in wastewater using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF MS) has been developed and validated. After solid-phase extraction with Cleanert PEP cartridges, sample extracts were analyzed by LC-QTOF MS for the simultaneous determination of levofloxacin, tetracycline, ketoprofen, meloxicam, lovastatin and fenofibrate in positive ion mode and chloramphenicol, diclofenac, and ibuprofen in negative ion mode. The separation and determination were performed on a C18 reversed-phase column (4.6 mm × 200 mm, 5 μm) under isocratic conditions. The pH value of the extracts, the type of SPE columns, and the MS conditions were optimized to increase the sample throughput and sensitivity. Quantitation was performed using external quantitation with standard solution mixtures. Under optimal conditions, the mean recovery for all the analytes from the water samples was more than 75.5%.

Gold nanoparticles for the quantification of very low levels of poly-diallyldimethylammonium chloride in river water by Bhekumuzi Gumbi; Jane Catherine Ngila; Patrick Gathura Ndungu (6963-6972).
We present a colorimetric method based on gold nanoparticle aggregation to detect and quantify poly-diallyldimethylammonium chloride (poly-DADMAC), a common water treatment polyelectrolyte, in river water. The protocol developed has an excellent linear range between 10 and 100 μg L−1 (R = 0.99), with a lower limit of detection of 0.54 μg L−1 and a lower limit of quantification of 1.5 μg L−1. The method has excellent intermediate precision (0.1–0.7%), relatively quick analysis times, requires no extraction or derivatization methods, and is robust and rugged. Results of spiked river water samples collected from the Umgeni River located in the province of KwaZulu-Natal, South Africa show that the method can detect low levels of poly-DADMAC in environmental matrices.

A convenient, robust and economical salting-out induced liquid–liquid microextraction (SILLME) method coupled with high-performance liquid chromatography/fluorescence detector (HPLC/FLD) for sample preparation, extraction and trace-level quantitative determination of six fluoroquinolones (FQs) in different samples was developed. The critical factors that influence the extraction efficiencies of the target analytes, such as the type of extraction solvent and salting-out reagent, the ratio of extraction solvent to salt, pH value and extraction time, were investigated. The system of acetonitrile/magnesium sulfate showed good extraction efficiencies for the target analytes. Under optimum conditions, the correlation coefficient (r2) was obtained within a range of 0.9990–0.9998 by spiking ultrapure water over a range of 0.002–0.100 μg mL−1. Excellent sensitivity was attained with limits of detection (LODs, S/N = 3) ranging from 0.07–0.41 ng mL−1, 0.09–0.62 ng mL−1, 0.48–2.49 μg kg−1, 0.80–5.00 ng mL−1, 0.78–5.58 ng mL−1 and 0.40–5.30 μg kg−1 for ultrapure water, field water, honey, milk, swine plasma and muscle, respectively. While precision with inter- and intra-day relative standard deviations (RSDs, n = 5) for ultrapure water were observed in the range of 0.4–4.0% and 1.3–6.8%, respectively. Finally, the developed method was successfully applied to all of the abovementioned matrices as a promising method for the analysis of FQs.

The electrocatalytic oxidation of two amino acids, l-arginine and l-lysine, and the purine base guanine at a copper nanoparticles-modified carbon paste electrode (NCPE) was investigated. The results were also compared with a copper microparticles-modified carbon paste electrode (MCPE). In the voltammograms recorded using these electrodes, one anodic oxidation peak related to the electrocatalytic oxidation of the analytes (guanine, l-arginine and l-lysine) appeared through an electrocatalytic mechanism (an electrochemical reaction followed by a chemical reaction regenerating the initial redox species, EC′). For the electrooxidation of guanine, l-arginine and l-lysine, the catalytic rate constants were obtained as 5.5, 2.98 and 0.56 cm3 mol−1 s−1, respectively, the transfer coefficients were obtained as 0.59, 0.43 and 0.46, respectively, and the diffusion coefficients were obtained as 1.93, 1.54 and 3.28 × 10−6 cm2 s−1, respectively. These analytes were oxidized on NCPE with higher rates at low potentials compared to MCPE. This behavior was related to the nanosize effect of copper nanoparticles. Finally, a simple, sensitive and time-saving amperometric procedure was developed for the analysis of the analytes in both batch and flow systems. Using the amperometric procedure, guanine, l-arginine and l-lysine were quantified with linear ranges of 12.5–62.5, 20–263 and 50–1100 μM, respectively, calibration sensitivities of 5.8, 4.8 and 4.3 × 10−2μA M−1, respectively, and limits of detection of 3.7, 4.3 and 8.6 μM, respectively, in batch system. In addition, for the flow system, guanine, l-arginine and l-lysine were quantified with linear ranges of 1–50, 50–700 and 100–1000 μM, respectively, calibration sensitivities of 4.4, 3.7 and 3.1 × 10−2μA M−1, respectively, and limits of detection of 0.23, 20.0 and 29.1 μM, respectively.

A europium-based fluorescence probe for detection of thiols in urine by Feng Xie; Hong Tan; Zhanbin Li; Hongbo Yang (6990-6996).
It is extremely necessary to develop a simple and sensitive analytical method for the detection of biothiols in biological fluids due to their vital biological functions. In this study, a fluorescent europium tetracycline complex (EuTc) was employed as a probe to detect biothiols, which is based on the enhancement and quenching of fluorescence of the EuTc complex by H2O2 and biothiols. After the addition of H2O2, significant enhancement in the fluorescence of EuTc can be observed due to the replacement of coordinated water molecules and the formation of a EuTc–H2O2 complex. In the presence of biothiols, however, the fluorescence of the EuTc complex was quenched due to the formation of disulfide bonds by H2O2. The fluorescence intensity of EuTc decreased linearly with increasing concentrations of biothiols. The detection limits for Cys, GSH and Hcys in aqueous solutions are 100 nM, 200 nM and 400 nM, respectively. The EuTc complex was successfully applied to detect total thiols in urine samples which showed a satisfactory result. Compared with other methods, the proposed method is advantageous because it provides a simple and low-toxicity detection procedure for thiols and possesses the ability to eliminate background fluorescence.

The redox behavior of a palladium(ii)-3,4-bis(2-hydroxyphenyl-imino) cyclobut-1-en-1,2-diol complex at Pt, Au and hanging mercury drop electrodes (HMDE) was studied for developing a low cost and precise method for determination of Pd concentration in road dust and other environmental samples. Hence, a controlled adsorptive accumulation of this complex on HMDE provided the basis for adsorptive cathodic stripping voltammetric (AdCSV) measurements of palladium at nanomolar levels at pH 9–10 and at −0.64 V vs. the Ag/AgCl reference electrode. The calibration plot was obtained in the range of 1.87 × 10−9 to 3.05 × 10−7 M (0.2 to 32.5 μg L−1) Pd. The limit of detection was found to be 4.70 × 10−10 M (0.05 μg L−1), with a relative standard deviation (RSD) of ±2.1% (n = 5) at 2.0 μg L−1 Pd level. Common anions and cations did not interfere in the determination of Pd concentration. The method was applied to the determination of the concentration of Pd in pure authentic samples, roadside dust and water samples. The method offers a simple system coupled with good reproducibility, accuracy, ruggedness and cost effectiveness.

To evaluate the glycoconjugated aroma compounds of three cultivars of tobacco, an enzymatic hydrolysis method followed by gas chromatography-mass spectroscopy (GC-MS) was successfully established using a series optimization approach. In the first step, two hydrolytic enzymes were compared and β-glucosidase was chosen. Then, the critical variables that affect the concentration of aglycons were searched by Plackett–Burman design (PBD) and further optimized by central composite design (CCD) of response surface methodology. The optimum conditions were: 60 h for hydrolysis time, 36.8 °C for hydrolysis temperature, 5.6 for pH, 2.1 mL for solvent volume, 2 mg mL−1 for β-glucosidase concentration and 200 rpm for agitation speed. Under the optimized experimental conditions, this method gave a satisfactory precision and linearity with relative standard deviation (RSD) less than 20% and Pearson correlation coefficients greater than 0.99. Finally, the proposed method was successfully employed to analyze flue-cured tobacco (FCT), sun-cured tobacco (SCT) and oriental tobacco (OT). Most of the aglycons were significantly different in the three cultivars and the corresponding characteristic components were discussed in detail.

A THF–water solvent system was used in the development of a method for the LC-MS/MS determination of chloramphenicol (CAP), thiamphenicol (TAP) and florfenicol (FFC) in bovine muscle. The tetrahydrofuran (THF)–water solvent system was recently demonstrated to possess superior figures of merit compared to either the methanol–water or acetonitrile–water solvent systems which are almost exclusively used in LC-MS for the determination of fenicols in food producing animals. The figures of merit included ease of de-solvation when electrospray ionization (ESI) is employed in the negative mode thus leading to more intense mass spectral signals. This phenomenon was shown to be due to greater association of the methyl groups in methanol or acetonitrile with the amide nitrogen common in all the three fenicols. This association is absent in a THF–water solvent system since it is devoid of methyl groups that can easily interact with the amide nitrogen. As a result of the use of the THF–water solvent system, the method detection limits (MDLs) were 0.047, 2.1 and 4.3 μg kg−1 for CAP, TAP and FFC respectively while the limits of quantitation were 0.141, 6.3 and 12.9 μg kg−1 for CAP, TAP and FFC respectively. The decision limits i.e. CCα values according to the Commission Decision 2002/657/EC criteria were 0.36, 50 and 111 μg kg−1 for CAP, TAP and FFC respectively. Linearities were also within acceptable values i.e. 0.9983, 0.9916 and 0.9996 for CAP, TAP and FFC respectively.

Genkwa flos, a traditional Chinese medicine, displays severe hepatotoxicity when it is excessively or chronically used in its raw form. It has been proven that the chloroform extracts within Genkwa flos are responsible for its hepatotoxicity. Note that the vinegar process procedure may weaken the toxicity and enhance the therapeutic effects. This study was conducted to investigate a quality control method of the chloroform extracts of Genkwa flos and to identify the potential hepatotoxic ingredients using HL-7702 cells. A LC-MS method was developed and fully validated to simultaneously determine three flavonoids (apigenin, genkwanin and hydroxygenkwanin), three lignans (syringaresinol, medioresinol and matairesinol) and two diterpene esters (yuanhuacine and genkwadaphnin) in the chloroform extracts. With satisfactory linearity, precision, repeatability, stability and recovery, the developed method was then applied to compare the content changes of the eight compounds in raw and processed herbs. After processing, the content of flavonoids increased, the lignans did not obviously change, while the diterpene esters decreased. Compared with the blank control group, the HL-7702 cells treated with the two diterpene esters exhibited obvious morphology change, viability decrease and increase in the level of hepatic marker enzymes in cell culture supernatant. The study showed a comprehensive quantitative method to simultaneously determine eight compounds of the chloroform extracts from Genkwa flos, and they indicated that yuanhuacine and genkwadaphnin could be two of the potential hepatotoxic substances of the herb.

An analytical method based on solid phase extraction (SPE) with XAD-2 for the enrichment of seven phthalate esters in light alcoholic drinks (alcohol content below 6% alc vol−1) and in soft drinks has been developed using gas chromatography coupled with an ion-trap mass spectrometer detector (GC/IT-MS). The instrumental analytical protocol was found to yield a linear calibration in the range of 1–800 ng mL−1 with r2 values ≥ 0.912; the limits of detection (LOD) vary between 0.2 pg μL−1 and 20 pg μL−1, whereas the limits of quantification (LOQ) range between 0.6 pg μL−1 and 41 pg μL−1 with relative standard deviations (RSDs) of ≤4.8 and ≤5.5, respectively. The interday precision ranges between 3.6 and 6.3%, whereas the intraday accuracy is between 7.9 and 12.4%. This simple, reliable, reproducible and low-cost analytical method has been applied to several commercial light alcoholic samples such as beers and soft drinks. DEP (0.1–1.0 ng mL−1), DiBP (0.2–2.5 ng mL−1), DBP (1.9–4.4 ng mL−1), BBP (0.08–0.8 ng mL−1), DEHP (3.6–101 ng mL−1) are present in every sample analyzed, whereas DMP (1.9 ng mL−1) and iBcEP (0.08 ng mL−1) are found only in one sample of beer.

In the present study, the electrochemical oxidation of atenolol (ATN) at gold nanoparticles (GNPs)/multiwall carbon nanotubes (MWCNTs)-modified glassy carbon electrode (GCE) was studied using cyclic voltammetry, differential pulse voltammetry and chronoamperometry. The synergetic effects of GNPs and MWCNTs highly improved electrochemical response and sensitivity of the sensor. The effect of experimental parameters, including amount of MWCNTs, GNPs, electrodeposition time, solution pH and scan rate on the sensor response, was evaluated. Under optimum experimental conditions, the respective linear calibration range, detection limit and sensitivity of the sensor were obtained as 1–60 μM, 0.5 μM and 350 nA μM−1 for differential pulse voltammetry and 0.1–1 μM, 59 nM and 4.405 μA μM−1 for chronoamperometry. The proposed sensor was successfully applied to the determination of atenolol in pharmaceutical samples, human urine and blood serum as real samples. The method was also applied to monitor ATN in urine samples of a dosage-received volunteer. Satisfactory recoveries of analyte from samples clearly revealed that the proposed sensor is well applicable to clinical analysis, quality control and a routine determination of drugs in pharmaceutical formulations.

Polypyrrole (PPy)-functionalized Fe3O4 magnetic nanoparticles (Fe3O4–PPy) were prepared and applied as magnetic solid-phase extraction (MSPE) sorbents for the extraction of polycyclic aromatic hydrocarbons (PAHs) from environmental water samples. To achieve the optimal extraction performance, several parameters were investigated, including the amounts of sorbent, volume of desorption solvent, extraction and desorption time, inorganic salt and organic solvent content of the sample matrix. Coupled with gas chromatography/mass spectrometry (GC/MS) analysis, a rapid, sensitive and cost-effective method for the analysis of PAHs in aqueous samples was established. The low detection limits (LODs) for the target PAHs in the proposed method were in the range of 0.38 to 5.01 ng L−1, which are much lower than those LODs reported in previous methods. Good linearity of the detection method was obtained with correlation coefficients (R) between 0.9904 and 0.9992. Satisfactory reproducibility was also achieved with relative standard deviations (RSDs) less than 13.3% by an intra- and inter-day precision evaluation. The recoveries of the 10 PAHs in several environmental water samples ranged from 72.4% to 115.7% with the RSDs being less than 9.7%.

The use of novel radially porous silica particles and gold thin film hybrid-based spectroscopy for the sensitive and anion-selective detection of As(iii) is described. The method is based on the selective formation of electrostatic complexes between As(iii) and an amine functionality on the porous silica particles and the effect of this on changes in the surface plasmon resonance (SPR) profiles of the gold thin film. Since silica particles have radially oriented mesopores, the sizes of which gradually increase from the center to the outer surface of the particle, they provide very large surface area, and permit target analytes to easily access active receptors within the pores. Thus, the amine-functionalized silica particles contain much higher concentration As(iii) receptors than conventional porous nanoparticles or a flat surface, which results in a lower detection limit. In addition, due to the surface properties of silica as a dielectric spacing layer, it allows the sensing platform to be more sensitive and stable. The SPR properties of the resulting selectively formed complexes are altered, leading to significant changes in SPR reflectance (ΔR) near the SPR angle. The limit of detection of the method was determined to be 1.0 nM, which is ca. 80 times more sensitive than the U.S. EPA regulation level (10 μg L−1≈ 130 nM). The response is essentially linear in the concentration range of 10–600 nM on a semi-log scale. The method also shows good selectivity for As(iii) in the presence of H2PO4, SO42−, NO3, and Cl and is feasible for use in the analysis of tap (drinking) water; therefore, it would be applicable for use in environmental and biological monitoring.

A fast method for simultaneous determination of amantadine, rimantadine, and chlorpheniramine in seven animal derived samples including pork, chicken, duck, pig liver, chicken liver, pig kidney, and egg was developed using liquid chromatography-tandem mass spectrometry, and employed a new multifunctional syringe filter that makes the cleanup procedure simple and rapid based on the QuEChERS (quick, easy, cheap, effective, rugged and safe) method. The method was validated using amantadine-d15, rimantadine-d4 and chlorpheniramine-d6 as internal standards for three analytes. Good linearities (R2 > 0.9938) were obtained over the concentration range from 2 μg L−1 to 200 μg L−1 for amantadine and rimantadine, and from 0.2 μg L−1 to 20 μg L−1 for chlorpheniramine. The precision was evaluated by intra- and inter-day assays and the relative standard deviations were all within 9.85%. Mean recoveries ranged from 89.9% to 105%. The limits of detection and quantification were 0.5 and 1.0 μg kg−1 for both amantadine and rimantadine, and 0.05 and 0.1 μg kg−1 for chlorpheniramine, respectively. The application of the developed method in real samples showed that amantadine and chlorpheniramine were respectively detected with percentages of 3.7% and 0.3% in all tested samples.

Considered a leading “green” separation technology, supercritical fluid chromatography (SFC) can be the best technique to separate and estimate chemical compounds. A precise, accurate and robust supercritical fluid chromatographic method was developed and validated for the determination of assay and uniformity of content of ramipril and telmisartan in their combined dosage forms. The chromatographic separation was achieved on a Zorbax SB-Phenyl column (150 mm × 4.6 mm, 5 μm) using supercritical carbon dioxide doped with 10% v/v methanol as a modifier, at a flow rate of 2.0 mL min−1. Column oven temperature was 35.0 °C, and UV detection was performed at 205 nm. The densities and polarities of the mobile phase were optimized from the effects of pressure, temperature and modifier concentration on chromatographic parameters. The developed method was validated in terms of specificity, linearity and range, accuracy, robustness and precision applying International Conference on Harmonization guidelines. The method was successfully applied for the assay and for the uniformity of content of different marketed formulations containing ramipril and telmisartan individually and in combination.

A rapid and sensitive LC-MS/MS analysis of diapocynin in rat plasma to investigate in vitro and in vivo pharmacokinetics by Hardik Chandasana; Yashpal S. Chhonker; Veenu Bala; Yarra Durga Prasad; Vishnu L. Sharma; Rabi S. Bhatta (7075-7082).
Natural products have been used traditionally for the treatment and prevention of diseases. Diapocynin is the analogue of the naturally occurring apocynin, a well-known constituent of Picrorhiza kurroa. It has antioxidant properties and also is effective in neuro-inflammatory diseases. To investigate in vitro and in vivo pharmacokinetics of diapocynin, a simple and sensitive method was developed and validated in rat plasma using high-performance liquid chromatography coupled with a mass spectrometer (LC-MS/MS). The developed and validated method requires low volume of the plasma (25 μL). The analytes were extracted by a simple protein precipitation method using acetonitrile. Chromatography resolution = n was achieved by using a Thermo Accucore C18 (5 μ, 4.6 × 150 mm) column using a mobile phase of acetonitrile and ammonium acetate buffer (10 mM, pH 4.0). The present method was successfully applied for in vitro clearance (using rat liver microsomes) and in vivo oral pharmacokinetic studies in the Sprague Dawley rats.

Multifunctional SERS substrates of Fe3O4@Ag2Se/Ag: construction, properties and application by Yun Wu; Hai Yang; Ling Zhu; Anjian Xie; Shikuo Li; Jiming Song; Yuhua Shen (7083-7087).
In this work, Fe3O4@Ag2Se/Ag nanocomposites were fabricated on a self-cleaning and recyclable surface-enhanced Raman scattering (SERS) substrate via a simple and ingenious precipitation shift reaction. The superparamagnetic property of Fe3O4 facilitates the efficient purification of products with the assistance of an external magnetic field. The as-prepared substrates have excellent stability and show no significant loss after six recycles for the photodegradation of rhodamine B (RhB). The unique Raman enhancement effect of Fe3O4@Ag2Se/Ag substrates indicates that RhB can be detected at a concentration as low as 1 × 10−8 M. Furthermore, the experimental results also suggest that these nanocomposites may be promising multifunctional SERS substrate candidates for environmental monitoring, cleaning and so on.

Back cover (7089-7090).