Analytical and Bioanalytical Chemistry (v.392, #7-8)

is the author or co-author of over 130 research articles, reviews, and book chapters. He is a member of the International Advisory Board of Analytical and Bioanalytical Chemistry. Dr Fetzer worked for over 20 years as an analytical chemist for the Chevron Corporation and now runs his own consulting company, Fetzpahs Consulting, in Hercules, CA, USA. His book ‘Career Management for Chemists—A Guide to Success in a Chemistry Career’, was published by Springer.

Kirk-Othmer Separation technology, 2nd ed. by John C. Fetzer (1253-1254).

Determination of ethanol in ionic liquids using headspace solid-phase microextraction–gas chromatography by Fei Zhao; Thehazhnan K. Ponnaiyan; Christa M. Graham; Constance A. Schall; Sasidhar Varanasi; Jared L. Anderson (1271-1275).
The application of ionic liquids (ILs) as nonderivatizing solvents for the pretreatment and regeneration of cellulose is a growing area of research. Here we report the development of a rapid and simple method for the determination of residual ethanol content in two hydrophilic ILs, 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium acetate. The method utilizes headspace solid-phase microextraction coupled with gas chromatography at elevated extraction temperatures, resulting in rapid equilibration times. The effect of IL water content on the ethanol extraction efficiency is presented. Recovery experiments carried out in real samples gave recoveries ranging from 96.8 to 98.2%. Figure Ionic liquids are becoming widely used as solvents for the regeneration of lignocellulosic biomass. In this communcation, a method based on headspace solid-phase microextraction coupled to gas chromatography is described to determine the ethanol content in two widely used ionic liquid matrices
Keywords: Ionic liquid; Room temperature ionic liquid; Ethanol; Gas chromatography; Solid-phase microextraction; Headspace extraction

Spatial distribution of heme species in erythrocytes infected with Plasmodium falciparum by use of resonance Raman imaging and multivariate analysis by Alois Bonifacio; Sara Finaurini; Christoph Krafft; Silvia Parapini; Donatella Taramelli; Valter Sergo (1277-1282).
The multivariate algorithm hierarchical cluster analysis is applied to sets of resonance Raman spectra collected from human erythrocytes infected with the malaria parasite Plasmodium falciparum. The images obtained yield information about the distribution of hemoglobin and hemozoin (or malaria pigment) within the parasitized cells and about their molecular structure. This method has the advantage of conveying more information than other imaging approaches based on resonance Raman spectroscopy, and it is a promising tool to study the hemozoin formation process and its interaction with antimalarial drugs within unstained, well-preserved parasites.
Keywords: Malaria; Plasmodium falciparum ; Hemozoin; Imaging; Resonance Raman; Multivariate analysis

Mercury speciation and total trace element determination of low-biomass biological samples by Vivien F. Taylor; Brian P. Jackson; Celia Y. Chen (1283-1290).
Current approaches to mercury speciation and total trace element analysis require separate extraction/digestions of the sample. Ecologically important aquatic organisms—notably primary consumers such as zooplankton, polychaetes and amphipods—usually yield very low biomass for analysis, even with significant compositing of multiple organisms. Individual organisms in the lower aquatic food chains (mussels, snails, oysters, silversides, killifish) can also have very low sample mass, and analysis of whole single organisms is important to metal uptake studies. A method for the determination of both methyl Hg and total heavy metal concentrations (Zn, As, Se, Cd, Hg, Pb) in a single, low-mass sample of aquatic organisms was developed. Samples (2 to 50 mg) were spiked with enriched with 201MeHg and 199Hg, then leached in 4 M HNO3 at 55 °C for extraction of MeHg. After 16 h, an aliquot (0.05 mL) was removed to determine mercury species (methyl and inorganic Hg) by isotope dilution gas chromatography inductively coupled plasma mass spectrometry (ICP-MS). The leachate was then acidified to 9 M HNO3 and digested in a microwave at 150 °C for 10 min, and total metal concentrations were determined by collision cell ICP-MS. The method was validated by analyzing five biological certified reference materials. Average percent recoveries for Zn, As, Se, Cd, MeHg, Hgtotal and Pb were 99.9%, 103.5%, 100.4%, 103.3%, 101%, 97.7%, and 97.1%, respectively. The correlation between the sum of MeHg and inorganic Hg from the speciation analysis and total Hg by conventional digestion of the sample was determined for a large sample set of aquatic invertebrates (n = 285). Excellent agreement between the two measured values was achieved. This method is advantageous in situations where sample size is limited, and where correlations between Hg species and other metals are required in the same sample. The method also provides further validation of speciation data, by corroborating the sum of the Hg species concentrations with the total Hg concentration.
Keywords: Mercury speciation; Trace metals; Species-specific isotope dilution; Low-mass samples; Inductively coupled plasma mass spectrometry; Acid leaching; Microwave digestion

Prospects for monolithic nano-LC columns in shotgun proteomics by Vilém Guryča; Sylvie Kieffer-Jaquinod; Jerôme Garin; Christophe D. Masselon (1291-1297).
We report a premier side-by-side comparison of two leading types of monolithic nano-LC column (silica-C18, polystyrene) in shotgun proteomics experiments. Besides comparing the columns in terms of the number of peptides from a real-life sample (Arabidopsis thaliana chloroplast) that they identified, we compared the monoliths in terms of peak capacity and retention behavior for standard peptides. For proteomics applications where the mobile phase composition is constrained by electrospray ionization considerations (i.e., there is a restricted choice of ion-pairing modifiers), the polystyrene nano-LC column exhibited reduced identification power. The silica monolith column was superior in all measured values and compared very favorably with traditional packed columns. Finally, we investigated the performances of the monoliths at high flow rates in an attempt to demonstrate their advantages for high-throughput identification.
Keywords: Liquid chromatography; Shotgun proteomics; Monoliths; Nano-LC columns; Peak capacity

Unraveling the metabolic transformation of tetrazepam to diazepam with mass spectrometric methods by Birthe Schubert; Marion Pavlic; Kathrin Libiseller; Herbert Oberacher (1299-1308).
The metabolic transformation pathways of the 1,4-benzodiazepine tetrazepam (C16H17ClN2O, average mass: 288.772) were studied with capillary LC-QqTOF-MS and -MS/MS by analyzing human plasma and urine samples collected from healthy volunteers. Each volunteer took 50 mg of tetrazepam, given in the form of one tablet of Myolastan (Sanofi-Synthelabo, Vienna, Austria). Accurate molecular mass measurements in full-scan mode (scan range: 50–700) were used to survey the collected samples for putative metabolic transformation products. Full-scan fragment ion mass spectra were collected in subsequent LC/MS/MS experiments. Each spectrum was matched to a spectral library containing 3759 MS/MS-spectra of 402 compounds, including eighteen different benzodiazepines, to prove the structural relatedness of a tentative metabolite to tetrazepam. This “similarity search” approach provided a rapid and powerful tool to exclude non-drug-related species, even without any knowledge of the fragmentation chemistry. Interpretation of tandem mass spectrometric data was only required in order to elucidate the site of transformation. Using this strategy, 11 major classes of tetrazepam metabolites were identified. Possible metabolic routes from tetrazepam to diazepam (C16H13ClN2O, average mass: 284.740) via repeated hydroxylation and dehydration of the cylohexenyl moiety were discovered. No evidence for extensive hydroxylation of tetrazepam at position 3 of the diazepine ring was found. In contrast to what is commonly believed, this distinct transformation reaction may be of only minor importance. Furthermore, the occurrence of demethylation, hydration, and glucuronidation reactions was proven. Figure Principle workflow applied for the identification of tetrazepam metabolites
Keywords: Mass spectrometry; Liquid chromatography; Electrospray ionization; Tetrazepam; Metabolism; Library search

This paper describes the fabrication of microarrays that enable the parallel electroporation of small interfering RNAs (siRNAs) into mammalian cells. To optimize the conditions of microarray preparation and electric pulsing, a self-assembled monolayer was formed on a gold electrode, and a cationic polymer was adsorbed by the entire surface of the monolayer. siRNA was then adsorbed by the cationically modified electrode through electrostatic interactions. Human embryonic kidney cells stably transformed with the expression construct of green fluorescent protein (GFP) were used to examine the electric pulse-triggered transfer of GFP-specific siRNA. A single electric pulse was applied to the cells cultured on the electrode at a field strength of 240 V cm−1. The expression of GFP was significantly suppressed in a sequence-specific manner two days after pulsing. Microscopic observation and flow-cytometric analysis revealed that the expression of GFP was attenuated in the majority of cells in a loading-dependent manner. Moreover, the effect of siRNA could be temporally controlled by changing the culture periods before pulsing. When a micropatterned self-assembled monolayer was used as a platform for loading siRNA in an array format, gene silencing was spatially restricted to the regions where specific siRNA was loaded. From these results, we conclude that array-based electroporation provides an excellent means of individual transfer of siRNAs into mammalian cells for high-throughput gene function studies. Figure Electroporation of siRNA on the microarray.
Keywords: Electroporation; Small interfering RNA; Electric pulse; Cell microarray; Functional genomics; Micropattern

A glass microfluidic chip for continuous blood cell sorting by a magnetic gradient without labeling by Bai-Yan Qu; Zhi-Yong Wu; Fang Fang; Zhi-Ming Bai; Dong-Zhi Yang; Shu-Kun Xu (1317-1324).
This paper presents a microfluidic chip for highly efficient separation of red blood cells (RBCs) from whole blood on the basis of their native magnetic properties. The glass chip was fabricated by photolithography and thermal bonding. It consisted of one inlet and three outlets, and a nickel wire of 69-μm diameter was positioned in the center of a separation channel with 149-μm top width and 73-μm depth by two parallel ridges (about 10 μm high). The two ridges were formed simultaneously during the wet etching of the channels. The nickel wire for generating the magnetic gradient inside the separation channel was introduced from the side of the chip through a guide channel. The external magnetic field was applied by a permanent magnet of 0.3 T placed by the side of the chip and parallel to the main separation channel. The RBCs were separated continuously from the 1:40 (v/v) diluted blood sample at a flow rate in the range 0.12–0.92 μL/min (9–74 mm/min) with the chip, and up to 93.7% of the RBCs were collected in the middle outlet under a flow rate of 0.23 μL/min. The cell sedimentation was alleviated by adjusting the specific density of the supporting media with bovine serum albumin. Quantum dot labeling was introduced for visual fluorescence tracking of the separation process. The uneven distribution phenomenon of the blood cells around the nickel wire was reported and discussed. Figure Glass chip with single Ni wire aligned in the microchannel for continuous magnetic separation of blood cells
Keywords: Microfluidic chip; Magnetic gradient; Blood sample; Red blood cells; Quantum dot labeling

The determination of glutathione-4-hydroxynonenal (GSHNE), E-4-hydroxynonenal (HNE), and E-1-hydroxynon-2-en-4-one (HNO) in mouse liver tissue by LC-ESI-MS by Molly M. Warnke; Eranda Wanigasekara; Sharad S. Singhal; Jyotsana Singhal; Sanjay Awasthi; Daniel W. Armstrong (1325-1333).
Glutathione (GSH) conjugation of 4-hydroxy-2(E)-nonenal (HNE) is an efficient means of cellular detoxification. HNE is a byproduct of lipid peroxidation which has shown toxicity but also signaling roles. E-1-hydroxynon-2-en-4-one (HNO) is another byproduct of lipid peroxidation which has the same molecular weight as HNE. This study presents the LC-MS detection of GS-HNE, HNE, and HNO in tissue samples without derivatization and with minimal sample preparation. Tissue samples were taken from wild-type mice and knock-out mice, which have been bred without the RLIP76 transfer protein. Extraction procedures were developed to determine GS-HNE and HNE levels in the mouse liver tissue. A gradient elution LC–MS method was developed for GS-HNE analysis using electrospray ionization and selected ion monitoring (SIM). The HNE/HNO method involves isocratic elution due to instability issues. Higher levels of GSHNE, HNE, and HNO were found in the knock-out animals, due to the absence of the RLIP76 transport mechanism.
Keywords: LC–MS; E-4-Hydroxynonenal (HNE); RLIP76; Glutathione-4-hydroxynonenal (GSHNE)

Screening of DHFR-binding drugs by MALDI-TOFMS by Paul Hannewald; Benoît Maunit; Jean-François Muller (1335-1344).
The class of antimetabolite chemotherapeutical agents has been used to treat cancers in humans for almost 50 years and gives significant results by binding dihydrofolate reductase (DHFR), a key enzyme in DNA synthesis. Therefore, finding new active compounds inhibiting DNA synthesis through their binding to DHFR is of prime interest. The aim of this work is to describe a protocol designed to study the binding of compounds to DHFR. This screening protocol involves matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) detection of target-bound compounds. Firstly, a screening protocol is developed and proves to be a simple, fast, and specific method to characterize the binding capability of a compound. Secondly, the possibility of determining the relative affinities of DHFR-binding compounds by comparing MALDI-TOFMS data is discussed. A ratio is calculated for a compound X such as $$R{left( X ight)} = frac{{A.I._{{denaturation}} {left( X ight)}}}{{A.I._{{direct}} {left( X ight)}}}$$ (where AIdirect and AIdenaturation are the average absolute intensities of a binding compound X before and after denaturation of DHFR). It is shown that by using this protocol, one can characterize the strength of the binding of different compounds. These two strategies are then applied to screen green tea (Camellia sinensis) extracts for DHFR-binding compounds, and epigallocatechin gallate is shown to be an active compound with a relative affinity between those of pyrimethamine and methotrexate.
Keywords: Dihydrofolate reductase; Dihydrofolate reductase binding drugs; Screening; Mass spectrometry; Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Methotrexate; Aminopterin

Among the various methods exploitable to determine the bioavailability of drugs, reversed-phase liquid chromatography (RPLC) appears to be suited to creation of patterns of prediction. In this context a new stationary phase was designed in this work to reproduce, in terms of chemical structure, as accurately as possible, the main elements of cellular membranes; which include phospholipids and cholesterol molecules. An efficient synthetic pathway was developed to prepare ligands that contain a phosphate head, a long alkyl chain chemically bonded to silica, and a cholesteric moiety, in order to mimic both hydrophilic and hydrophobic interactions, and “membrane-like” organization, respectively. The new stationary phase was characterized by Fourier-transform infra red (FTIR) and 1H–13C, 1H–31P, and 1H–29Si cross-polarization magic-angle-spinning nuclear magnetic resonance (CP MAS NMR) spectroscopy. Its chromatographic behavior has been studied by classical classification tests for RPLC columns. Despite its low surface coverage, the material produced exhibits high shape selectivity, possibly due to the organization of the grafted moieties.
Keywords: HPLC; CP MAS NMR; Phospho–cholestric phase; Shape selectivity; Methylene selectivity; Hydrophobicity

Stereoselective recognition of bilirubin and biliverdin by poly(l-lysine) (PLL), poly(d-lysine) (PDL), and poly(l-arginine) (PLA) and their micelles with dodecanoate ions (C12) at different pH has been studied using a combination of vibrational and electronic circular dichroism. Biliverdin has been found to be more sensitive to pH in its complexes with the polypeptides. In acidic media in the complexes with PLL–C12 and PDL–C12 the conformation becomes more closed than the characteristic one found at physiological pH. Partial flattening and chiral self-association of bilirubin molecules takes place at higher concentrations with PLL and PDL. For both pigments, inversions of the ECD signals are observed in the systems with PLA at pH ≥ 8.5. This study was carried out in order to clarify the role of Lys and Arg residues in pigment binding to serum albumin. The circular dichroism spectra obtained for bilirubin bound to different mammalian serum albumins have been compared with the homology within the IIA principal ligand-binding structural domains. Analysis suggests that the chiroptical properties of the pigment in the complexes with serum albumins depend on the location of Lys and/or Arg at positions 222 and 199 in the binding site. Figure Analysis of circular dichroism spectra suggests that the chiroptical properties and the spatial structure of the bile pigments in the complexes with serum albumin depend on the location of Lys and/or Arg at positions 222 and 199 in the binding site of protien
Keywords: Bilirubin; Biliverdin; Polypeptide binding; Serum albumin; CD spectra; Stereoselective recognition

Near-infrared spectroscopy (NIRS) has been widely used in the pharmaceutical field because of its ability to provide quality information about drugs in near-real time. In practice, however, the NIRS technique requires construction of multivariate models in order to correct collinearity and the typically poor selectivity of NIR spectra. In this work, a new methodology for constructing simple NIR calibration models has been developed, based on the spectrum for the target analyte (usually the active principle ingredient, API), which is compared with that of the sample in order to calculate a correlation coefficient. To this end, calibration samples are prepared spanning an adequate concentration range for the API and their spectra are recorded. The model thus obtained by relating the correlation coefficient to the sample concentration is subjected to least-squares regression. The API concentration in validation samples is predicted by interpolating their correlation coefficients in the straight calibration line previously obtained. The proposed method affords quantitation of API in pharmaceuticals undergoing physical changes during their production process (e.g. granulates, and coated and non-coated tablets). The results obtained with the proposed methodology, based on correlation coefficients, were compared with the predictions of PLS1 calibration models, with which a different model is required for each type of sample. Error values lower than 1–2% were obtained in the analysis of three types of sample using the same model; these errors are similar to those obtained by applying three PLS models for granules, and non-coated and coated samples. Based on the outcome, our methodology is a straightforward choice for constructing calibration models affording expeditious prediction of new samples with varying physical properties. This makes it an effective alternative to multivariate calibration, which requires use of a different model for each type of sample, depending on its physical presentation.
Keywords: NIR spectroscopy; API determination; Correlation coefficients; PLS calibration

Aluminium malate complexes have a high relevance in biological systems. The anionic species present in an aqueous aluminium malate mixture can be investigated by ion chromatography coupled online with inductively coupled plasma atomic emission spectroscopy. As malic acid is a chiral ligand, the experiments were carried out using the racemic and the enantiopure forms. In both systems four anionic complexes are observable in the model solutions. One of two crystallized and well-characterized anions, [Al4Malate6]6− and [Al4Malate4]2−, serves as a reference anion for the assignment of one of the species occurring in the model solutions. The main species in the enantiopure aluminium malate model with biological relevance over a wide pH range is the [Al4Malate4]2− anion. The [Al4Malate6]6− anion is not present in the racemic model solution. This anion suffers complete species disintegration after dissolution. The kinetics of the decay is first order and the activation energy of the decomposition is 74 kJ/mol. A retention model for ion chromatography was used for the determination of the effective charges of the species. The effective charge obtained by the retention model was calibrated in relation to nominal charges of the anions by using several well-defined and differently charged anions and anionic aluminium species.
Keywords: Speciation; Ion chromatography; Aluminium malate complexes; Charge determination; Reference materials

Determination of glycolic acid in cosmetics by online liquid chromatography–Fourier transform infrared spectrometry by J. Kuligowski; A. Breivogel; G. Quintás; S. Garrigues; M. de la Guardia (1383-1389).
An isocratic online liquid chromatography Fourier transform infrared procedure has been developed for the determination of glycolic acid in cosmetics. The method involves the ultrasound-assisted extraction of glycolic acid from the samples with an acetonitrile:phosphate buffer (25 mM, pH 2.7) (3:97 v/v). The extracts were centrifuged and filtered before their injection into the chromatography system, which was equipped with a C18 column and used a flow rate of 150 μL min−1. FTIR spectra were acquired using a time-resolved rapid scan mode. To calculate the chromatograms, the spectral area was integrated between 1288 and 1215 cm−1, with baseline correction established between 1319 and 1150 cm−1, after correcting for the eluent spectral background. Peak area values of the extracted sample chromatograms were interpolated from an external calibration curve. The method provided a limit of detection of 0.034 mg mL−1 and a relative standard deviation of 6% for five measurements at the 0.174 mg mL−1 concentration level. Recovery values obtained by spiking 400 mg of three commercially available samples with amounts of glycolic acid from 3.7 to 9.8 mg ranged between 99.6 and 101%. The results obtained for the commercial samples agree well with their declared concentrations. An attempt to directly determine glycolic acid by attenuated total reflectance measurements using partial least squares calibration showed that results were strongly influenced by compounds coextracted from the matrix.
Keywords: Glycolic acid; Cosmetics analysis; Online liquid chromatography Fourier transform infrared; Attenuated total reflectance (ATR); Partial least squares (PLS)

Determination of critical micelle concentration with the rotating sample system by Linus T. Kao; Gautam N. Shetty; Miklós Gratzl (1391-1396).
A novel experimental approach using the rotating sample system (RSS) is proposed here for the determination of the critical micelle concentration (CMC) of surfactants. The RSS has been conceived in our laboratory as a convection platform for physicochemical studies and analyses in microliter-sized sample drops. The scheme allows for vigorous rotation of the drop despite its small size through efficient air–liquid mechanical coupling. Thus, changes in surface properties of aqueous samples result in corresponding modulation of the hydrodynamic performance of the RSS, which can be utilized to investigate interfacial phenomena. In this work, we demonstrate that the RSS can be used to study the effects of surfactants on the surface and in the bulk of very small samples with hydrodynamic electrochemistry. Potassium ferrocyanide is employed here with cyclic voltammetry to probe the air–water interface of solutions containing Triton X-100. The CMC of this surfactant determined using this approach is 140 ppm, which agrees well with reported values obtained with conventional methods in much larger samples. The results also demonstrate that besides the CMC, variations in bulk rheological properties can also be investigated in very small specimens using the RSS with a simple method.
Keywords: Cyclic voltammetry; Micelles; Surfactants; Rotating sample system; Hydrodynamic electrochemistry

Implementation of terbium-sensitized luminescence in sequential-injection analysis for automatic analysis of orbifloxacin by E. J. Llorent-Martínez; P. Ortega-Barrales; A. Molina-Díaz; A. Ruiz-Medina (1397-1403).
Orbifloxacin (ORBI) is a third-generation fluoroquinolone developed exclusively for use in veterinary medicine, mainly in companion animals. This antimicrobial agent has bactericidal activity against numerous Gram-negative and Gram-positive bacteria. A few chromatographic methods for its analysis have been described in the scientific literature. Here, coupling of sequential-injection analysis and solid-phase spectroscopy is described in order to develop, for the first time, a terbium-sensitized luminescent optosensor for analysis of ORBI. The cationic resin Sephadex-CM C-25 was used as solid support and measurements were made at 275/545 nm. The system had a linear dynamic range of 10–150 ng mL−1, with a detection limit of 3.3 ng mL−1 and an R.S.D. below 3% (n = 10). The analyte was satisfactorily determined in veterinary drugs and dog and horse urine.
Keywords: Orbifloxacin; Sequential-injection analysis; Optosensor; Urine; Veterinary drug

Polymers imprinted with PAH mixtures—comparing fluorescence and QCM sensors by Peter A. Lieberzeit; Konstantin Halikias; Adeel Afzal; Franz L. Dickert (1405-1410).
Molecular imprinting with binary mixtures of different polycyclic aromatic hydrocarbons (PAH) is a tool for design of chemically highly sensitive layers for detection of these analytes. Sensor responses increase by one order of magnitude compared with layers imprinted with one type of template. Detection limits, e.g. for pyrene, reach down to 30 ng L−1 in water, as could be observed with a naphthalene and pyrene-imprinted polyurethane. Comparing sensor characteristics obtained by QCM and fluorescence reveals different saturation behaviours indicating that, first, single PAH molecules occupy the interaction centres followed by gradual excimer incorporation at higher concentrations finally leading to substantial quenching, when all accessible cavities are occupied. The plateau in the mass-sensitive measurements suggests that up to 80% of the cavities generated in the MIP are re-occupied. Displacement measurements between chrysene and pyrene revealed that for imprinted layers with very high pyrene sensitivities the signals of both PAH are additive, whereas in materials with lower pyrene uptake the two analytes replace each other in the interaction sites of the polymer.
Keywords: PAH detection; Chemical sensors; Molecular imprinting; Fluorescence; QCM

Design of acidochromic dyes for facile preparation of pH sensor layers by Gerhard J. Mohr; Heidrun Müller; Beate Bussemer; Annegret Stark; Tommaso Carofiglio; Sabine Trupp; Rüdiger Heuermann; Thomas Henkel; Daniel Escudero; Leticia González (1411-1418).
Eight new acidochromic dyes have been synthesised that can be used for optically monitoring pH in the range from 3 to 12. Their corresponding pK a values have been both measured and calculated theoretically by means of density functional theory. The synthesis of these new dyes is facile without the need for chromatographic purification. The dyes can be covalently linked to polymers containing hydroxyl functions such as cellulose, polyurethane hydrogel, and hydroxyalkyl methacrylate. The resulting sensor layers exhibit significant colour changes both in the UV and in the visible spectral range. Figure Sensor layers based on covalently immobilised pH indicator dyes show fast and fully reversible colour changes.
Keywords: Optical sensors; pH measurement; Polymers

Synthesis and binding site characteristics of 2,4,6-trichlorophenol-imprinted polymers by Qin-Zhong Feng; Li-Xia Zhao; Bao-Lin Chu; Wei Yan; Jin-Ming Lin (1419-1429).
2,4,6-Trichlorophenol (2,4,6-TCP)-imprinted micro- and submicrospheres prepared by precipitation polymerization were compared with templated materials obtained by conventional bulk polymerization. The influence of the type and amount of functional monomer, the type and amount of cross-linker, polymerization temperature, porogen, and the ratio of template molecule and functional monomer to cross-linker on the size of the obtained particles were investigated. UV–Vis spectrophotometer experiments revealed that the microsphere polymers provided higher affinity to the template in contrast to imprinted polymers prepared by bulk polymerization. The binding properties of the microspheres, including binding isotherms and affinity distribution, were studied via Freundlich isotherm affinity distribution (FIAD) analysis. The obtained results indicated that microspheres prepared by precipitation polymerization provided superior rebinding properties during equilibrium binding in contrast to bulk polymers and submicrosphere polymers. Moreover, release experiments showed that 80% of rebound 2,4,6-TCP was released from the imprinted microspheres within the first 2 h, while more intimately bound 2,4,6-TCP molecules were released in the following 40 h. The morphologies and porosities of the resulting imprinted materials were characterized by scanning electron microscopy (SEM) and Brunauer–Emmett–Teller (BET) analysis, respectively. The microsphere polymers exhibited a regular spherical shape with a high degree of monodispersity to the corresponding bulk polymers. Furthermore, the micro- and submicrospheres were characterized by narrow distribution of pores in contrast to a heterogeneity index of m = 0.6647 for the microsphere imprinted polymer. Nitrogen adsorption BET isotherms of imprinted submicrospheres ( ), control submicrospheres ( ), imprinted microspheres (■),control microspheres ( ), particulate bulk imprinted polymer ( ), and particulate bulk control polymer ( )
Keywords: Molecularly imprinted polymers; Precipitation polymerization; Imprinted microsphere; Imprinted submicrosphere; 2,4,6-Trichlorophenol

The synthesis and evaluation of a molecularly imprinted polymer (MIP) as a selective matrix solid-phase dispersion (MSPD) sorbent, coupled with high-performance liquid chromatography for the efficient determination of chloramphenicol (CAP) in fish tissues are studied. The polymer was prepared using CAP as the template molecule, vinylpyridine as the functional monomer and ethylene glycol dimethacrylate as the cross-linking monomer, and sodium dodecyl sulfate as the surfactant in the presence of water as a solvent by miniemulsion polymerization. The CAP-imprinted polymers and nonimprinted polymers (NIPs) were characterized by Fourier transform IR spectroscopy, scanning electron microscopy, and static adsorption experiments. The CAP-imprinted material prepared showed high adsorption capacity, significant selectivity, and good site accessibility. The maximum static adsorption capacity of the CAP-imprinted and the NIP material for CAP was 78.4 and 59.9 mg g-1, respectively. The relative selectivity factors of this CAP-imprinted material were larger than 1.9. Several parameters influencing the MSPD process were optimized. Finally, the CAP-imprinted polymers were used as the sorbent in MSPD to determine CAP in three kinds of fishes and resulted in satisfactory recovery in the range 89.8–101.43%. CAP-imprinted polymer as a sorbent in MSPD is better than C18 and attapulgite in terms of both recovery and percent relative standard deviation. The baseline noise was measured from a chromatogram of a blank fish sample which was treated after the MSPD procedure using CAP-imprinted polymer as a sorbent. Signal values of 3 times the noise (signal-to-noise ratio of 3) and 10 times the noise (signal-to-noise ratio of 10) were used to calculate the limit of detection and the limit of quantitation of the calibration curve. The limit of detection for CAP was 1.2 ng g-1 and the limit of quantitation was 3.9 ng g-1.
Keywords: Molecularly imprinted polymers; Matrix solid-phase dispersion; Chloramphenicol; High-performance liquid chromatography

Ionic liquids as mobile phase additives for the high-performance liquid chromatographic analysis of fluoroquinolone antibiotics in water samples by Antonio V. Herrera-Herrera; Javier Hernández-Borges; Miguel Ángel Rodríguez-Delgado (1439-1446).
In this work, four ionic liquids differing in the length of the alkyl chain on the imidazolium cation and one ionic liquid containing tetraethylammonium, all with the same counterion, (i.e. 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIm-BF4), 1-butyl-3-methylimidazolium tetrafluoroborate (BMIm-BF4), 1-hexyl-3-methylimidazolium tetrafluoroborate (HMIm-BF4), 1-methyl-3-octylimidazolium tetrafluoroborate (MOIm-BF4), and tetraethylammonium tetrafluroborate (Et4N-BF4)) were tested as mobile phase additives for HPLC separation of a group of seven basic fluoroquinolone (FQ) antibiotics for human and veterinary use (i.e. fleroxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, and difloxacin) using a conventional reversed-phase Nova-Pak C18 column. Fluorescence detection was used. Among the ionic liquids selected, use of BMIm-BF4 enabled effective separation of these compounds with relatively low analysis time (14 min). The best separation was achieved by isocratic elution at 1 mL min−1 with 5 mmol L−1 BMIm-BF4 and 10 mmol L−1 ammonium acetate at pH 3.0 with 13% (v/v) acetonitrile. Limits of detection (LODs) for fluorescence detection were in the range 0.5–11 μg L−1. The method was tested by analyzing several water samples after the optimization of a suitable solid-phase extraction (SPE) procedure using Oasis HLB cartridges. Mean recovery values were above 84% for all analytes with LODs in the range 1–29 ng L−1.
Keywords: High-performance liquid chromatography; Fluorescence detection; Fluoroquinolones; Antibiotics; Ionic liquids; Water

A straightforward quantification method is presented for the application of asymmetric flow field-flow fractionation (AsFlFFF) combined with inductively coupled plasma mass spectrometry (ICPMS) to the characterization of colloid-borne metal ions and nanoparticles. Reproducibility of the size calibration and recovery of elements are examined. Channel flow fluctuations are observed notably after initiation of the fractionation procedure. Their impact on quantification is considered by using 103Rh as internal reference. Intensity ratios measured for various elements and Rh are calculated for each data point. These ratios turned out to be independent of the metal concentration and total sample solution flow introduced into the nebulizer within a range of 0.4–1.2 mL min−1. The method is applied to study the interaction of Eu, U(VI) and Th with a mixture of humic acid and clay colloids and to the characterization of synthetic nanoparticles, namely CdSe/ZnS-MAA (mercaptoacetic acid) core/shell-coated quantum dots (QDs). Information is given not only on inorganic element composition but also on the effective hydrodynamic size under relevant conditions. Detection limits (DLs) are estimated for Ca, Al, Fe, the lanthanide Ce and the natural actinides Th and U in colloid-containing groundwater. For standard crossflow nebulizer, estimated values are 7 × 103, 20, 3 × 102, 0.1, 0.1 and 7 × 10−2 µg L−1, respectively. DLs for Zn and Cd in QD characterization are 28 and 11 µg L−1, respectively. Figure Outline of the principle of AsFlFFF/ICPMS and its application to the characterization of colloidal species in groundwater systems
Keywords: Flow field-flow fractionation; Inductively coupled plasma mass spectrometry; Radionuclides; Colloids; Humic acids; Nanoparticles; Quantum dots; Quantification; Hydrodynamic size

Determination of higher carboxylic acids in snow samples using solid-phase extraction and LC/MS-TOF by Matthias Kippenberger; Richard Winterhalter; Geert K. Moortgat (1459-1470).
The objective of this work was to develop a method to determine the concentrations of higher organic acids in snow samples. The target species are the homologous aliphatic α,ω-dicarboxylic acids from C5 to C13, pinonic acid, pinic acid and phthalic acid. A preconcentration procedure utilizing solid phase extraction was developed and optimized using solutions of authentic standards. The influences of different parameters such as flow rate during extraction and the concentration of the eluent on the efficiency of the extraction procedure were investigated. The compounds of interest were separated by HPLC and detected by a quadrupole time-of-flight mass spectrometer (qTOF-MS). The recovery rate (extraction efficiency) of the extraction procedure was found to vary between 41% for tridecanedioic acid and 102% for adipic acid. The limits of detection were determined for all compounds and were between 0.9 nmol/L (dodecanedioic acid) and 29.5 nmol/L (pinonic acid). An exception is pinic acid, for which a considerably higher detection limit of 103.9 nmol/L was calculated. Snow samples were collected in December 2006 and January 2007 at the Fee glacier (Switzerland) from locations at heights from 3056 to 3580 m asl and from different depths within the snow layer. In total, the analysis of 61 single snow samples was performed, and the following compounds could be quantified: homologous aliphatic α,ω-dicarboxylic acids with 5–12 carbon atoms and phthalic acid. Tridecanedioic acid, pinonic and pinic acid were identified in the samples but were not quantified due to their low concentrations. The three most abundant acids found in the molten snow samples were glutaric acid (C5-di; 3.90 nmol/L), adipic acid (C6-di; 3.35 nmol/L) and phthalic acid (Ph; 3.04 nmol/L).
Keywords: Dicarboxylic acids; Snow samples; Solid-phase extraction; Pinonic acid; Phthalic acid; Fee glacier

Simultaneous extraction of several persistent organic pollutants in sediment using focused ultrasonic solid-liquid extraction by A. Errekatxo; A. Prieto; O. Zuloaga; A. Usobiaga; N. Etxebarria; L. A. Fernández (1471-1478).
Focused ultrasonic solid-liquid extraction (FUSLE) has been optimised for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), phthalate esters (PEs), and nonylphenols (NPs) in sediment samples. Optimisation was performed using naturally polluted freeze-dried sediment samples. The variables studied during the optimisation process were: percentage of maximum power (10–60%), extraction time (10–300 s), number of cycles (1–9), composition of the extraction solvent (acetone-n-hexane, 10:90–90:10), and sample mass (0.1–1 g). The volume of the extractant was constant (10 mL) and the extraction was performed at 0°C in an ice-bath during the optimisation process. All these variables were studied using an experimental design approach by means of The Unscrambler software. The extraction time and the operational variables (number of cycles and power) had no statistically significant effect in the extraction and they were held at 2 min, 20% power, and seven cycles, respectively. The mass and the addition of non-polar solvent (n-hexane) had a negative effect in the extraction yield and, thus, the mass was held at 0.5 g and pure acetone was used as extraction solvent. After those variables were optimised, the effect of the extraction temperature (0°C or room temperature) was also studied. The validation of the extraction method was performed using NIST-1944 reference material in the case of PAHs and PCBs. Because no certified reference sediment is available for PEs and NPs, the results obtained for FUSLE were compared with those obtained for microwave-assisted extraction (MAE) under conditions optimised elsewhere. In all the cases the analysis were performed by gas chromatography-mass spectrometry (GC-MS). Good accuracy were achieved in all cases. The limits of detection (LODs) obtained were between 0.10 and 1.70 ng g−1 for PAHs (except for naphthalene 5.33 ng g−1), 0.02 and 0.16 ng g−1 for PCBs, 46 and 188 ng g−1 for PEs, and 0.6 and 12.4 μg g−1 for NPs. The precision was around 5–10% for most of the PAHs and PCBs and around 2–10% for most of the PEs and NPs.
Keywords: Focused ultrasonic solid-liquid extraction (FUSLE); Optimisation; Experimental design; Sediment samples; Persistent organic pollutants

Derivative Fourier transform infrared (FTIR) spectroscopy and curve fitting have been used to investigate the effect of a thermal treatment on the ν 1 ν 3 PO4 domain of modern bones. This method was efficient for identifying mineral matter modifications during heating. In particular, the 961, 1022, 1061, and 1092 cm−1 components show an important wavenumber shift between 120 and 700 °C, attributed to the decrease of the distortions induced by the removal of CO3 2− and HPO4 2− ions from the mineral lattice. The so-called 1030/1020 ratio was used to evaluate crystalline growth above 600 °C. The same analytical protocol was applied on Magdalenian fossil bones from the Bize–Tournal Cave (France). Although the band positions seem to have been affected by diagenetic processes, a wavenumber index—established by summing of the 961, 1022, and 1061 cm−1 peak positions—discriminated heated bones better than the 1030/1020 ratio, and the splitting factor frequently used to identify burnt bones in an archaeological context. This study suggest that the combination of derivative and curve-fitting analysis may afford a sensitive evaluation of the maximum temperature reached, and thus contribute to the fossil-derived knowledge of human activities related to the use of fire.
Keywords: Derivative FTIR; Curve fitting; Archaeological burnt bones; Diagenetic processes; Hydroxyapatite

NIR-FT-Raman spectroscopic analytical characterization of the fruits, seeds, and phytotherapeutic oils from rosehips by Carlos Eduardo da Silva; Peter Vandenabeele; Howell G. M. Edwards; Luiz Fernando Cappa de Oliveira (1489-1496).
In this study nondestructive Raman spectroscopic analysis of rosehips has been conducted by laser excitation at 1064 nm, with the objective of direct measurement of different parts of the fruit, including the inside and outside of the seeds, while preserving the integrity of the more representative chemicals. Carotenoid substances are responsible for the major Raman features in the spectra of the fruit parts; analysis of the ν 1 mode (1520 cm−1) strongly indicates the presence of a C9 carotene, and analysis of the main characteristic carotene band set (C = C, C–C, and C–CH3 stretching modes at 1520, 1157, and 1007 cm−1, respectively) suggests the presence of β-carotene as the main constituent. Raman spectra of the seed parts show the presence of unsaturated fatty acids, which are predominant inside the seed; these spectra also reveal the fatty products content comprises cis isomers. Analysis of the CH-stretching region bands and comparison with those in the spectra obtained from linoleic acid and commercial rosehip oil indicate that the relative band intensity of the CH-stretching mode is strongly affected by the chemical environment of the fatty acid esters present in both parts (inside and outside) of rosehips seeds.
Keywords: Rosehips; Rosaceae family; Phytotherapeutic oils; FT-Raman spectra; Carotenoids

Uncertainty estimation in organic elemental analysis using information from proficiency tests by R. Companyó; R. Rubio; A. Sahuquillo; R. Boqué; A. Maroto; J. Riu (1497-1505).
We evaluate the uncertainty in organic elemental analysis of C, H, N, and S. We use data from six proficiency tests (PTs), in which some 35 Spanish laboratories participated. The uncertainty of the technique is estimated from the relative within-laboratory and between-laboratory variances for pure substances and samples with complex matrices (soil, powdered milk, oil, ash, and petroleum coke). We also calculate the relative standard uncertainties for individual laboratories when analysing pure substances using historical data from the participation of each laboratory in different editions of PTs. The uncertainty values obtained for the individual laboratories are comparable with the uncertainty of the technique and correlate with the combined z-scores. The evolution over time of those laboratories participating in common editions of PTs is also evaluated.
Keywords: Organic elemental analysis; Proficiency testing schemes; Uncertainty evaluation

Preparation of reference material for organochlorine pesticides in a herbal matrix by Yiu Chung Wong; Siu Kay Wong; Tat Ting Kam (1507-1513).
The development of reference material for four organochlorine pesticides, namely hexachlorobenzene and three isomers of hexachlorocyclohexane (α-hexachlorocyclohexane, β-hexachlorocyclohexane and γ-hexachlorocyclohexane), in a ginseng root sample is presented. Raw materials (Panax ginseng) were purchased from a local market and confirmed to contain certain levels of incurred organochlorine pesticide residues by a validated gas chromatography-mass selective detection method. A total of more than 300 bottles each containing 25 g of samples were prepared after the materials had been freeze-dried, milled and thoroughly mixed. The homogeneity and stability of samples from randomly selected bottles were verified and the reference values were characterized using a highly precise isotope dilution gas chromatography-mass spectrometry (ID-GCMS) method that was recently developed by our laboratory. The purity of standard organochlorine chemicals was determined against certified reference materials to establish the accuracy of the ID-GCMS analysis. The concentrations (± expanded uncertainty) of hexachlorobenzene, α-hexachlorocyclohexane, β-hexachlorocyclohexane and γ-hexachlorocyclohexane in the reference material were 0.198 ± 0.015, 0.450 ± 0.022, 0.213 ± 0.011 and 0.370 ± 0.032 mg kg−1, respectively. A portion (70 bottles) of the samples was also used in a proficiency testing (PT) scheme for assessing the testing capabilities of field laboratories. The consensus mean values of the PT obtained from the 70 participants were on the same order but deviated by −2.7 to −14.1% from those of the assigned reference values. Because of the wide spread of participants’ data (relative standard deviation ranging from 44 to 56%), the PT results were not included in the calculation of the assigned values of the reference materials. The materials served as suitable reference materials to ascertain the quality control and validation processes for the determination of organochlorine pesticides in herbal matrices.
Keywords: Reference material; Organochlorine pesticides; Isotope dilution mass spectrometry; Proficiency testing programme