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Analytical and Bioanalytical Chemistry (v.386, #5)

Revising the quantitative analysis laboratory: What to keep? What to change? by Cynthia K. Larive (pp. 1191-1194).

is Professor of Analytical Chemistry at the University of California Riverside. She has an active research program involving bioanalytical and environmental analytical applications of NMR Spectroscopy (for more information see INTER-REF locator="http://www.chem.ucr.edu/faculty/larive/larive.html" locator-type="URL"http://www.chem.ucr.edu/faculty/larive/larive.html/INTER-REF ). Professor Larive is also active in curricular reform and the promotion of undergraduate research. She is editor-in-chief and principal investigator of the Analytical Sciences Digital Library INTER-REF locator="http://www.asdlib.org" locator-type="URL"http://www.asdlib.org/INTER-REF), an Internet-based resource for instructors, students and practitioners of analytical chemistry. This digital library is a collection of peer-reviewed websites on topics including pedagogical approaches, analytical techniques, applications and classroom resources.

Quality assurance challenge 4 by Manfred Reichenbächer; Jürgen W. Einax (pp. 1195-1195).

Solution to geometric chemistry challenge by Juris Meija; William B. Jensen (pp. 1197-1197).

Optical biochemical and chemical sensors – Europt(r)ode VIII by Günter Gauglitz (pp. 1199-1200).

is Professor at the Eberhard-Karls-University of Tübingen, working in analytical and physical chemistry. He is chairman of the GDCh Division of Analytical Chemistry and chaired the Europt(r)ode VIII meeting. For the last ten years his main scientific interests have centred on research and development in the field of chemical and biochemical sensors, with special focus on the characterisation of interfaces of polymers and biomembranes by surface spectroscopic techniques, the use of spectral interferometry to monitor changes in the optical thickness of thin layers, and the effects of Fresnel reflectivity at interfaces.

New chromogenic and fluorogenic reagents and sensors for neutral and ionic analytes based on covalent bond formation–a review of recent developments by Gerhard J. Mohr (pp. 1201-1214).

To date, hydrogen bonding and Coulomb, van der Waals and hydrophobic interactions are the major contributors to non-covalent analyte recognition using ionophores, ligands, aptamers and chemosensors. However, this article describes recent developments in the use of (reversible) covalent bond formation to detect analyte molecules, with special focus on optical signal transduction. Several new indicator dyes for analytes such as amines and diamines, amino acids, cyanide, formaldehyde, hydrogen peroxide, organophosphates, nitrogen oxide and nitrite, peptides and proteins, as well as saccharides have become available. New means of converting analyte recognition into optical signals have also been introduced, such as colour changes of chiral nematic layers. This article gives an overview of recent developments and discusses response mechanisms, selectivity and sensitivity.

Keywords: Chemosensor; Optical probe; Optical sensor; Biomolecules; Chemical warfare agents

A simplified measurement procedure and portable electronic photometer for disposable sensors based on ionophore-chromoionophore chemistry for potassium determination by A. Palma; A. Lapresta-Fernández; J. M. Ortigosa-Moreno; M. D. Fernández-Ramos; M. A. Carvajal; L. F. Capitán-Vallvey (pp. 1215-1224).

A field-portable photometer for potassium determination with disposable sensors has been developed. It can be applied to routine water and beverage analysis. The disposable sensor is based on ionophore-chromoionophore chemistry. A colour change in the sensing film is detected by measuring the transmitted intensity with a solid state photodetector. Optical excitation at 660 nm is emitted by a light-emitting diode (LED). Negative feedback for LED bias and thermal correction were included to improve system stability. Additionally, a measurement procedure is presented, characterized and validated for in situ photometer use and real-time results. This simplified procedure is based on prior preparation of the disposable sensor in its acidic form and on the use of an absorbance ratio as analytical parameter. The only requirement for analysis is prior equilibration with a buffered sample solution for 3 min and absorbance measurement before and after equilibration. Good sensitivity in the concentration range 5 μM to 100 mM and very good repetitively and stability were achieved that are comparable to those obtained with bulkier analytical instrumentation. Given the compact size, low weight, rapid response and low energy requirement of the electronic photometer developed here, this measurement system is suitable for potassium determination in the field.

Keywords: Field portable instrument; Optical chemical sensor; Photometer; Ionophore-chromoionophore chemistry; Potassium determination; Water and beverage analysis

Emission-based optical carbon dioxide sensing with HPTS in green chemistry reagents: room-temperature ionic liquids by Ozlem Oter; Kadriye Ertekin; Derya Topkaya; Serap Alp (pp. 1225-1234).

We describe the characterization of a new optical CO₂ sensor based on the change in the fluorescence signal intensity of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) in green chemistry reagents—room-temperature ionic liquids (RTILs). As far as we are aware, this is the first time RTILs, 1-methyl-3-butylimidazolium tetrafluoroborate (RTIL-I) and 1-methyl-3-butylimidazolium bromide (RTIL-II), have been used as matrix materials with HPTS in an optical CO₂ sensor. It should be noted that the solubility of CO₂ in water-miscible ionic liquids is approximately 10 to 20 times that in conventional solvents, polymer matrices, or water. The response of the sensor to gaseous and dissolved CO₂ has been evaluated. The luminescence intensity of HPTS at 519 and 521 nm decreased with the increasing concentrations of CO₂ by 90 and 75% in RTIL-I and RTIL-II, respectively. The response times of the sensing reagents were in the range 1–2 min for switching from nitrogen to CO₂, and 7–10 min for switching from CO₂ to nitrogen. The signal changes were fully reversible and no significant hysteresis was observed during the measurements. The stability of HPTS in RTILs was excellent and when stored in the ambient air of the laboratory there was no significant drift in signal intensity after 7 months. Our stability tests are still in progress.

Keywords: Room temperature ionic liquids (RTILs); Carbon dioxide; Optical sensor; HPTS

Fluorescence sensor using a molecularly imprinted polymer as a recognition receptor for the detection of aluminium ions in aqueous media by Sing Muk Ng; R. Narayanaswamy (pp. 1235-1244).

This paper describes the investigation of a molecularly imprinted polymer (MIP) as a sensing receptor for Al³⁺ ion detection by using an optical approach. Al³⁺ ion was adopted as the template molecule and 8-hydroxyquinoline sulfonic acid ligand as the fluorescence tag. The polymer was synthesised using acrylamide as monomer, 2-hydroxyethyl methacrylate as co-monomer and ethylene glycol dimethracylate as cross-linker. The free radical polymerisation was performed in methanol and initiated by 2,2′-azobisisobutyronitrile at 70 °C. The imprinted polymer was fluorometrically characterised using a fibre optic attachment in a self-designed flow-cell. NaF was used to leach the Al³⁺ ion from the MIP. The optimum pH for the rebinding of Al³⁺ ion with the leached polymer was found to be pH 5 and the fluorescence response was found to be stable within the buffer strength range of 0.05–0.10 M. The fluorescence intensity during Al³⁺ ion rebinding was inversely dependent on temperature, and a low interference response (<3%) toward metal ions except for Cu²⁺ and Zn²⁺ ions was observed. The polymer rebinding repeatability study conducted over 9 cycles with Al³⁺ ion (0.8×10⁻⁴ M) was found to give an RSD value of 2.82% with a standard deviation of 0.53. The dynamic range of the system was found to be linear up to 1.0×10⁻⁴ M Al³⁺ ion with a limit of detection of 3.62 μM.

Keywords: Molecularly imprinted polymer (MIP); Optical sensor; Aluminium ion; 8-Hydroxyquinoline sulfonic acid (8HQS); Fluorescence complex

A novel luminescent lifetime-based optrode for the detection of gaseous and dissolved oxygen utilising a mixed ormosil matrix containing ruthenium (4,7-diphenyl-1,10-phenanthroline)₃Cl₂ (Ru.dpp) by P. Roche; R. Al-Jowder; R. Narayanaswamy; J. Young; P. Scully (pp. 1245-1257).

A novel luminescent lifetime optrode is presented for the detection of gaseous and dissolved oxygen. The optrode utilises ruthenium (4,7-diphenyl-1,10-phenanthroline)₃Cl₂ as the sensing fluorophore immobilised in a hydrophobic ormosil matrix. The ormosil matrix is synthesised at room temperature from octyltriethoxysilane and methyltriethoxysilane precursors. Investigations of different ormosils were conducted and the most effective one was selected for optrode production. Optrodes were tested for responses to gaseous and dissolved oxygen. Their responses were modelled using traditional two-site or two-exponential methods and feed-forward artificial neural networks. Comparison of the two modelling methodologies is presented and further improvements in modelling and ormosil design are suggested.

Keywords: Optrode; Ormosil; Ruthenium (4,7-diphenyl-1,10-phenanthroline)₃Cl₂ ; Artificial neural networks; Oxygen

Single-detector micro-electro-mechanical scanning grating spectrometer by M. Kraft; A. Kenda; A. Frank; W. Scherf; A. Heberer; T. Sandner; H. Schenk; F. Zimmer (pp. 1259-1266).

A compact, robust grating spectrometer based on an optimised micro-electro-mechanical grating mirror component has been developed, built, and characterised. The application of an oscillating reflection grating micro-mirror component as scanning dispersive element in a modified Czerny–Turner monochromator layout enables the design of compact grating spectrometers capable of acquiring full spectra using a single detector element. Designed for a wavelength range between 1200 and 1900 nm, the spectrometer features a spectral resolution of 10 nm with wavelength stability better than ±0.5 nm. One-hundred scan spectra can be acquired in less than one second, or spectral changes can be monitored at time a resolution of less than 10 ms. In combination with a fibre-optic interface and a typical weight of less than 1 kg, this makes this novel type of fully portable micro-electro-mechanical near-IR scanning spectrometer an interesting alternative to existing spectrometers and opens a range of new applications, in particular the detection of major and minor components in the near-IR. MEMS SG spectrometer prototype

Keywords: Spectroscopy instrumentation; Miniaturised grating spectrometer; MEMS; Near-infrared; Rapid scanning

Development and characterization of an integrated silicon micro flow cytometer by R. Bernini; E. De Nuccio; F. Brescia; A. Minardo; L. Zeni; P. M. Sarro; R. Palumbo; M. R. Scarfi (pp. 1267-1272).

This paper describes an innovative integrated micro flow cytometer that presents a new arrangement for the excitation/detection system. The sample liquid, containing the fluorescent marked particles/cells under analysis, is hydrodynamically squeezed into a narrow stream by two sheath flows so that the particles/cells flow individually through a detection region. The detection of the particles/cells emitted fluorescence is carried out by using a collection fiber placed orthogonally to the flow. The device is based on silicon hollow core antiresonant reflecting optical waveguides (ARROWs). ARROW geometry allows one to use the same channel to guide both the sample stream and the fluorescence excitation light, leading to a simplification of the optical configuration and to an increase of the signal-to-noise ratio. The integrated micro flow cytometer has been characterized by using biological samples marked with standard fluorochromes. The experimental investigation confirms the success of the proposed microdevice in the detection of cells.

Keywords: Microfluidics; Microfabrication; Optical sensors; Biochips; High-throughput screening

Optical biochemical sensor for determining hydroperoxides in nonpolar organic liquids as archetype for sensors consisting of amphiphilic conetworks as immobilisation matrices by Michael Hanko; Nico Bruns; Joerg C. Tiller; Jürgen Heinze (pp. 1273-1283).

This paper reports the successful design of a prototype of an optical biochemical sensor for the determination of hydroperoxides in nonpolar organic liquids. The sensor consists of a matrix of an amphiphilic polymer conetwork (APCN), a novel class of very promising polymeric materials for easy preparation of biochemical sensor matrices. APCNs are characterised by nanoscopic phase separation between the hydrophilic and the hydrophobic phases. For medium ratios of conetwork composition, the domains of both phases are interconnected both on the surface of the conetworks and throughout the bulk. The APCNs have peculiar swelling properties—the hydrophilic phase swells in hydrophilic media and the hydrophobic phase swells in hydrophobic media. In both types of media dissolved reagents can diffuse from the solution into the swollen phase of the polymeric conetwork. This enables loading of the hydrophilic phase of the APCNs with enzymes and indicator reagents by simple impregnation. Hydrophobic analytes can diffuse into the polymeric conetwork via its hydrophobic phase and react with indicator reagents immobilised in the hydrophilic phase at the huge internal interface between the two opposite phases.To prepare the described hydroperoxide-sensitive biosensors, we used APCN films consisting of 58% (w/w) poly(2-hydroxyethyl acrylate) (PHEA) as hydrophilic chains and 42% (w/w) polydimethylsiloxane (PDMS) as hydrophobic linkers. Horseradish peroxidase (HRP) and diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as indicator reagent were co-immobilised in this optically clear and transparent matrix. In this feasibility study the conditions investigated were principally those relevant to characterisation of the innovative matrix material and the disposable biosensor produced from it; the biosensor was not optimised. Sensitivity toward tert-butylhydroperoxide (tBuOOH) dissolved in n-heptane was acceptable, between approximately 1 and at least 50 mmol L⁻¹, even in the dry state. The response time was 1.7 to 5.0 min. No leaching of immobilised reagents was observed during a period of at least one hour. Pre-swelling the sensors with water increased the reaction rate and the total turnover number of the enzyme. In a dry atmosphere at 4 °C the sensors were found to be stable for at least two weeks.

Keywords: Nanophase-separated amphiphilic conetwork; Enzyme immobilisation; Optical biosensor; Nonpolar organic media; Horseradish peroxidase; Peroxide determination

Stacked films immobilization of MBTH in nafion/sol-gel silicate and horseradish peroxidase in chitosan for the determination of phenolic compounds by Jaafar Abdullah; Musa Ahmad; Lee Yook Heng; Nadarajah Karuppiah; Hamidah Sidek (pp. 1285-1292).

The stacked-film immobilization of 3-methyl-2-benzothiazolinone hydrazone (MBTH) in hybrid nafion/sol-gel silicate film and horseradish peroxidase (HRP) in chitosan, performed in order to allow the determination of phenolic compounds, was investigated via an optical method. The stacked films were deposited onto a microscope glass slide by a spin-coating technique. The quinone or free radical product formed by the enzymatic reactions of phenolic compounds interacts with MBTH to form azo-dye products, which can be measured spectrophotometrically at a wavelength of 500 nm. The color intensity of the product was found to increase in proportion to the phenolic concentration after 5 min of exposure. The response of the biosensor was linear over concentration ranges of 0.025–0.500, 0.010–0.070 and 0.050–0.300 mM for guaiacol, resorcinol and o-cresol, respectively, and gave detection limits of 0.010, 0.005 and 0.012 mM. The sensor exhibited good sensitivity and stability for at least two months.

Keywords: Optical biosensor; Horseradish peroxidase (HRP); Hybrid nafion/sol-gel silicate; Phenolic compounds

Fiber-optic flow-through sensor for online monitoring of glucose by Alen Pasic; Hans Koehler; Lukas Schaupp; Thomas R. Pieber; Ingo Klimant (pp. 1293-1302).

A new microdialysis-based glucose-sensing system with an integrated fiber-optic hybrid sensor is presented. Design and dimensions of the cell are adapted for its coupling with commercially available microdialysis techniques, thereby providing a new system for continuous glucose monitoring. The glucose level is detected via oxygen consumption which occurs as a consequence of enzymatic reaction between immobilized glucose oxidase and glucose. The use of gas-permeable Tygon tubing ensures complete and constant air-saturation of the measuring fluid in the cell. Nevertheless, a reference oxygen optode is used to detect and to compensate response changes caused by events like bacterial growth, temperature fluctuations, or failure of the peristaltic pump. In contrast to widely used electrochemical sensors, the response of the microdialysis-based fiber-optic glucose sensor is highly selective, making this sensor approach particularly advantageous for continuous glucose monitoring of patients in intensive care units. The effects of flow rate, pH, temperature, and common interferences on the sensor response are presented and discussed in detail. The sensor is evaluated in vitro using a 3-day continuous test in glucose-spiked plasma. The ability to measure glucose in humans is demonstrated by coupling the flow-through cell and commercially available microdialysis catheter CMA60. A 24-h monitoring test using this setup is successfully applied to a healthy volunteer.

Keywords: Continuous glucose monitoring; Glucose biosensor; Oxygen optode; Hybrid sensor; Microdialysis

A microchip sensor for calcium determination by P. Caglar; S. A. Tuncel; N. Malcik; J. P. Landers; J. P. Ferrance (pp. 1303-1312).

A newly designed glass-PDMS microchip-based sensor for use in the determination of Ca²⁺ ions has been developed, utilizing reflectance measurements from arsenazo III (1,8-dihydroxynaphthalene-3,6-disulfonic acid-2,7-bis[(azo-2)-phenyl arsenic acid]) immobilized on the surface of polymer beads. The beads, produced from cross-linked poly(p-chloromethylstyrene) (PCMS), were covalently modified with polyethylenimine (PEI) to which the Arsenazo III could be adsorbed. The maximum amount of Arsenazo III which could be immobilized onto the PEI-attached PCMS beads was found to be 373.71 mg g⁻¹ polymer at pH 1. Once fabricated, the beads were utilized at the detection point of the microfluidic sensor device with a fiber optic assembly for reflectance measurements. Samples were mobilized past the detection point in the sensor where they interact with the immobilized dye. The sensor could be regenerated and re-used by rinsing with HCl solution. The pH, voltage, linear range, and the effect of interfering ions were evaluated for Ca²⁺ determination using this microchip sensor. At the optimum potential, 0.8 kV, and pH 9.0, the linear range of the microchip sensor was 3.57 × 10⁻⁵ – 5.71 × 10⁻⁴ M Ca²⁺, with a limit of detection (LOD) of 2.68 × 10⁻⁵ M. The microchip biosensor was then applied for clinical analysis of calcium ions in serum with good results.

Keywords: Microchip sensor; Reflectance detection; Fiber optic technology; Calcium determination; Arsenazo III

Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay by Dhesingh Ravi Shankaran; Toshikazu Kawaguchi; Sook Jin Kim; Kiyoshi Matsumoto; Kiyoshi Toko; Norio Miura (pp. 1313-1320).

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl–keyhole limpet hemocyanin (TNPh–KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh–KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.

Keywords: TNT; SPR; Immunoassay; Indirect competitive immunoreaction; Antibody; Physical adsorption

Reversible immobilization of proteins with streptavidin affinity tags on a surface plasmon resonance biosensor chip by Yong-Jin Li; Li-Jun Bi; Xian-En Zhang; Ya-Feng Zhou; Ji-Bin Zhang; Yuan-Yuan Chen; Wei Li; Zhi-Ping Zhang (pp. 1321-1326).

Dissociation of biotin from streptavidin is very difficult due to their high binding affinity. The re-use of streptavidin-modified surfaces is therefore almost impossible, making devices containing them (e.g. surface plasmon resonance (SPR) sensor chips) expensive. This paper describes a new protocol for reversible and site-directed immobilization of proteins with streptavidin affinity tags on the streptavidin-coated SPR biosensor chip (SA chip). Two streptavidin affinity tags, nano-tag and streptavidin-binding peptide (SBP tag), were applied. They both can specifically interact with streptavidin but have weaker binding force compared to the biotin–streptavidin system, thus allowing association and dissociation under controlled conditions. The SA chip surface could be regenerated repeatedly without loss of activity by injection of 50 mM NaOH solution. The fusion construct of a SBP tag and a single-chain antibody to mature bovine prion protein (scFv-Z186-SBP) interacts with the SA chip, resulting in a single-chain-antibody-modified surface. The chip showed kinetic response to the prion antigen with equilibrium dissociation constant K _D≈4.01×10⁻⁷. All results indicated that the capture activity of the SA chip has no irreversible loss after repeated immobilization and regeneration cycles. The method should be of great benefit to various biosensors, biochips and immunoassay applications based on the streptavidin capture surface.

Keywords: Surface plasmon resonance; Streptavidin affinity tag; Nano-tag; Site-directed immobilization; Regeneration

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications by Tsuyoshi Nakayama; Yasunori Kurosawa; Satoshi Furui; Kagan Kerman; Masaaki Kobayashi; S. Ramachandra Rao; Yuji Yonezawa; Kouichi Nakano; Akihiro Hino; Shohei Yamamura; Yuzuru Takamura; Eiichi Tamiya (pp. 1327-1333).

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (μTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the “initial start-up” in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with μTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.

Keywords: Microfluidics; Air bubbles; Quantitative continuous-flow PCR

Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization by Katie A. Edwards; Antje J. Baeumner (pp. 1335-1343).

A novel protocol for the synthesis of dye-encapsulating liposomes tagged with DNA oligonucleotides at their outer surface was developed. These liposomes were optimized for use as signal enhancement agents in lateral-flow sandwich-hybridization assays for the detection of single-stranded RNA and DNA sequences. Liposomes were synthesized using the reverse-phase evaporation method and tagged with oligonucleotides by adding cholesteryl-modified DNA probes to the initial lipid mixture. This resulted in a greatly simplified protocol that provided excellent control of the probe coverage on the liposomes and cut the preparation time from 16 hours to just 6 hours. Liposomes were prepared using probe concentrations ranging from 0.00077 to 0.152 mol% of the total lipid, several hydrophobic and polyethylene glycol-based spacers between the cholesteryl anchor and the probe, and liposome diameters ranging from 208 nm to 365 nm. The liposomes were characterized by dynamic light scattering, visible spectroscopy, and fluorescence spectroscopy. Their signal enhancement functionality was compared by using them in lateral-flow optical biosensors for the detection of single-stranded DNA sequences. In these assays, an optimal reporter probe concentration of 0.013 mol%, liposome diameter of 315 nm, and liposome optical density of 0.4–0.6 at 532 nm were found. The spacer length between the cholesteryl anchor and the probe showed no significant effect on the signals in the lateral-flow assays. The results presented here provide important data for the general use of liposomes as labels in analytical assays, with specific emphasis on nucleic acid detection via lateral flow assays.

Keywords: Liposomes; DNA; Sandwich hybridization; Conjugation; Detection; Biosensor

Identification of in vitro metabolites of JWH-015, an aminoalkylindole agonist for the peripheral cannabinoid receptor (CB₂) by HPLC-MS/MS by Qiang Zhang; Peng Ma; Richard B. Cole; Guangdi Wang (pp. 1345-1355).

The in vitro microsomal metabolism of JWH-015, a ligand that exhibits a high binding affinity at the peripheral cannabinoid receptor CB₂, has been studied. A total of 22 metabolites were identified and structurally characterized. The metabolites are products of: 1) monohydroxylation on the naphthalene ring (m/z 344, M20 and M21), indole ring (m/z 344, M17 and M18), or the N-alkyl group (m/z 344, M14); 2) arene oxidation leading to dihydrodiols (m/z 362, M12 and M15); 3) dihydroxylation on the naphthalene ring (m/z 360, M7) or indole ring (m/z 360, M13), resulting from a combination of monohydroxylations on both the naphthalene and indole rings (m/z 360, M16), or a combination of monohydroxylations on the naphthalene ring and on the N-propyl group (m/z 360, M9); 4) trihydroxylation (m/z 378, M1, M3, M4, M6, and M10); 5) N-dealkylation (m/z 286, M19); 6) N-dealkylation and monohydroxylation on the naphthalene ring (m/z 302, M11); 7) N-dealkylation and dihydrodiol formation from arene oxidation (m/z 320, M2 and M5); 8) dehydrogenation after monohydroxylation on the N-alkyl group (m/z 326, M22); 9) dehydrogenation and monohydroxylation on the indole ring (m/z 342, M8).

Keywords: JWH-015; Metabolites; CB₂ agonist; HPLC-tandem mass spectrometry

Non-metal element detection by radio-frequency glow-discharge optical-emission spectrometry (rf-GD-OES) for determination of sol–gel-immobilized nucleotides by Tim M. Brewer; R. Kenneth Marcus (pp. 1357-1365).

A new method is presented for qualitative and quantitative determination of non-metal elements present in organic analyte species immobilized in a sol–gel matrix. Nucleic acids were chosen as well-defined relevant biomolecules for which element ratios could be used for detection and identification. Solid, lyophilized powders of ribose-form nucleotides (AMP, ADP, and ATP) were immobilized/entrapped in a methlytrimethoxysilane (mTMOS) sol–gel matrix and monitored for P (I) 214.9 nm, C (I) 193.0 nm, O (I) 130.2, and N (I) 149.3 nm emission by radio-frequency glow-discharge optical-emission spectroscopy (rf-GD-OES). Using the sol–gel method, analytical blanks were obtained by use of un-doped sol–gels. Empirical formula calculations by use of P (I) and C (I), P (I) and N (I), and P (I) and O (I) emission response ratios demonstrate the versatility of the technique as an element and species-specific detector. Results show there is high correlation between phosphorus and oxygen emission responses and the extent of phosphorylation, demonstrating the capacity of the method to produce vital qualitative and quantitative information for the specific nucleotide. Absolute sub-nanogram detection limits were achieved for all the elements studied.

Keywords: Sol–gel; Radio-frequency glow-discharge optical-emission spectroscopy; Nucleic acids; Phosphorylation

Comparing a selenium accumulator plant (Brassica juncea) to a nonaccumulator plant (Helianthus annuus) to investigate selenium-containing proteins by Sandra Mounicou; Anne P. Vonderheide; Jodi R. Shann; Joseph A. Caruso (pp. 1367-1378).

Selenoproteins have been identified in a diverse range of organisms, including bacteria and animals. Their occurrence and role in the plant kingdom are, however, less well-understood. This work investigated the water-soluble selenium-containing proteins extracted from a selenium-accumulating plant species (Brassica juncea) and a nonaccumulator species (Helianthus annuus) exposed to varying forms and concentrations of selenium. Firstly, protein extracts were analyzed by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry; specific detection was achieved by monitoring characteristic isotopes. Then, proteolytic digests of the plant extracts were analyzed by reversed phase chromatography coupled to ICP–MS in order to investigate selenoamino acid and selenopeptide content. Selenomethionine was observed to be the primary constituent of the proteins of the nonaccumulator plant, while selenocystine and selenomethionine were found in the same proportion in the accumulator extract. One main selenium-containing species was present at higher levels in the root digests than in the leaf digests; levels were greater in the nonaccumulator than in the accumulator plant.

Keywords: Brassica juncea (Indian mustard); Heliantus annuus (sunflower); Water-soluble selenium-containing proteins; HPLC/ICP–MS

Detection of bacteria by surface-enhanced Raman spectroscopy by Atanu Sengupta; Mirna Mujacic; E. James Davis (pp. 1379-1386).

The detection and identification of dilute bacterial samples by surface-enhanced Raman spectroscopy has been explored by mixing aqueous suspensions of bacteria with a suspension of nanocolloidal silver particles. An estimate of the detection limit of E. coli was obtained by varying the concentration of bacteria. By correcting the Raman spectra for the broad librational OH band of water, reproducible spectra were obtained for E. coli concentrations as low as approximately 10³ cfu/mL. To aid in the assignment of Raman bands, spectra for E. coli in D₂O are also reported. Figure Light scattering apparatus used to detect bacteria

Keywords: Bacteria detection; Bacterial pathogens ; Raman spectroscopy

Fluorescent detection of peptides and amino acids for capillary electrophoresis via on-line derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole by Hongyi Zhang; Isabelle Le Potier; Claire Smadja; Jiyou Zhang; Myriam Taverna (pp. 1387-1394).

An in-capillary derivatization of amino acids and peptides with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was developed for their subsequent capillary electrophoretic analysis with laser-induced fluorescence detection (λ _ex=488 nm). The in-capillary derivatization was achieved in zone-passing mode by introducing successive plugs of sample and NBD-F into a fused silica capillary previously equilibrated with an alkaline borate buffer. To prevent NBD-F hydrolysis and to achieve a reliable derivatization, NBD-F was prepared daily in absolute ethanol and a plug of absolute ethanol was introduced between the sample and NBD-F reagent plugs. Various parameters influencing the derivatization efficiency were investigated and the optimum conditions were as follows: background electrolyte (BGE), 20 mM borate buffer (pH 8.8); introduction time, 4 s for sample and 2 s for NBD-F; molar ratio of NBD-F/sample, above 215; temperature, 45 °C for amino acids and 35 °C for peptides; applied voltage, +15 kV. The validation of the in-capillary derivatization method under optimal conditions showed a good linearity between the heights of the derivative peaks and the concentrations of the amino acids. The intra-day relative standard deviations of the migration times and the peak heights were less than 1.3% and 4.6%, respectively. The efficient derivatization and separation of a mixture of valine, alanine, glutamic acid and aspartic acid were achieved using this technique. Peptides such as buccaline and β-protein fragment 1–42 could also be derivatized using the developed in-capillary derivatization procedure. In‑capillary derivatization and separation of amino acids with different concentrations. From the top to bottom the concentrations are 1.11×10⁻⁵ M, 5.55×10⁻⁶ M, 2.78×10⁻⁶ M, 6.95×10⁻⁷ M. for valine; 1.26×10⁻⁵ M, 6.30×10⁻⁶ M, 3.15×10⁻⁶ M, 7.88×10⁻⁷ M for alanine; 3.78×10⁻⁵ M, 1.89×10⁻⁵ M, 9.45×10⁻⁶ M, 2.36×10⁻⁶ M for glutamic acid;, 4.27×10⁻⁵ M, 2.14×10⁻⁵ M, 1.07×10⁻⁵ M, 2.68×10⁻⁶ M for aspartic acid. Experiment conditions: injection order: 4s for sample, 1s for absolute ethanol, and then 2s for 5.24×10⁻² M NBD‑F; BGE: 20 mM borate pH 8.77; Applied voltage: 15 kV.

Keywords: In-capillary derivatization; Capillary electrophoresis; Laser-induced fluorescence; 4-Fluoro-7-nitro-2,1,3-benzoxadiazole; Amine

Determination of fluoroacetic acid in water and biological samples by GC-FID and GC-MS in combination with solid-phase microextraction by Nadezhda L. Koryagina; Elena I. Savelieva; Natalia S. Khlebnikova; Nikolay V. Goncharov; Richard O. Jenkins; Andrey S. Radilov (pp. 1395-1400).

A novel procedure has been developed for determination of fluoroacetic acid (FAA) in water and biological samples. It involves ethylation of FAA with ethanol in the presence of sulfuric acid, solid-phase microextraction of the ethyl fluoroacetate formed, and subsequent analysis by GC-FID or by GC-MS in selected-ion-monitoring mode. The detection limits for FAA in water, blood plasma, and organ homogenates are 0.001 μg mL⁻¹, 0.01 μg mL⁻¹, and 0.01 μg g⁻¹, respectively. The determination error at concentrations close to the detection limit was less than 50%. For analysis of biological samples, the approach has the advantages of overcoming the matrix effect and protecting the GC and GC-MS systems from contamination. Application of the approach to determination of FAA in blood plasma and organ tissues of animals poisoned with sodium fluoroacetate reveals substantial differences between the dynamics of FAA accumulation and clearance in rabbits and rats.

Keywords: SPME; GC-MS; Fluoroacetate; Fluoroacetic acid; Ethylation; Fluorocitrate

A novel competitive fluorescence immunoassay for the determination of dibutyl phthalate by Ming-Cui Zhang; Qiong-E Wang; Hui-Sheng Zhuang (pp. 1401-1406).

A novel, sensitive, and specific competitive fluorescence immunoassay has been developed for the quantitative determination of dibutyl phthalate (DBP) using an antibody-coated plate format. Hapten was synthesized in order to produce polyclonal antibodies against dibutyl phthalate. Polyclonal antisera to dibutyl phthalate were generated in rabbits and used to construct the fluorescence immunoassay for measurement of dibutylphthalate. The assay had a detection limit of about 0.02 μg L⁻¹, a dynamic range of approximately 0.1–300 μg L⁻¹. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. The study demonstrated that the developed antiserum and fluorescence immunoassay procedure can be used to detect dibutyl phthalate in environmental samples such as tap water, river water, drinking water, and leachate from plastic drinking water bottles.

Keywords: Dibutyl phthalate; Determination; Hapten; Antibody; Fluorescence immunoassay

Methylmercury determination in biological samples using electrothermal atomic absorption spectrometry after acid leaching extraction by Mohammad Saber-Tehrani; Hamid Hashemi-Moghaddam; Mohammad Hadi Givianrad; Parviz Abroomand-Azar (pp. 1407-1412).

An efficient and sensitive method for the determination of methylmercury in biological samples was developed based on acid leaching extraction of methylmercury into toluene. Methylmercury in the organic phase was determined by electrothermal atomic absorption spectrometry (ETAAS). The methylmercury signal was enhanced and the reproducibility increased by formation of certain complexes and addition of Pd-DDC modifier. The complex of methylmercury with DDC produced the optimum analytical signal in terms of sensitivity and reproducibility compared to complexes with dithizone, cysteine, 1,10-phenanthroline, and diethyldithiocarbamate. Method performance was optimized by modifying parameters such as temperature of mineralization, atomization, and gas flow rate. The limit of detection for methylmercury determination was 0.015 μg g⁻¹ and the RSD of the whole procedure was 12% for human teeth samples (n=5) and 15.8% for hair samples (n=5). The method’s accuracy was investigated by using NIES-13 and by spiking the samples with different amounts of methylmercury. The results were in good agreement with the certified values and the recoveries were 88–95%.

Keywords: Methylmercury; Acid leaching; Teeth; ETAAS; DDC

Electrocatalytic reduction of hydrogen peroxide on modified graphite electrodes: application to the development of glucose biosensors by T. Dodevska; E. Horozova; N. Dimcheva (pp. 1413-1418).

The electrocatalytic activities of a series of compact graphites modified with microquantities of platinum metals (Pd or Pt+Pd) towards the electrochemical reduction of hydrogen peroxide were characterised. Operational parameters such as the optimal working potential, the influence of temperature and the resulting electrode characteristics were examined. The benefits of using graphite modified with Pt+Pd (mixture ratio 30%:70%) as the basic transducer in a glucose biosensor with improved sensitivity were demonstrated. It was proven that, under the working conditions chosen, the selected electrode (whether bare or covered with an enzyme layer) did not respond to any glutathione, uric acid or ascorbic acid (which all normally occur in biological fluids) present.

Keywords: Modified graphite electrodes; Hydrogen peroxide; Electroreduction; Glucose biosensor

Experimental parameters affecting sensitivity and specificity of a yeast assay for estrogenic compounds: results of an interlaboratory validation exercise by Willem Dhooge; Katrien Arijs; Isabel D’Haese; Sabrina Stuyvaert; Bram Versonnen; Colin Janssen; Willy Verstraete; Frank Comhaire (pp. 1419-1428).

In vitro assays are considered as the first step in a tiered approach to compound screening for hormonal activity. Although many new assays have been developed in recent years, little attention has been paid towards assay validation. Our objective was to identify critical experimental parameters in a yeast estrogen screen (YES) that affect its sensitivity and specificity. We investigated the role of incubation time, solvent type, yeast inoculum growth stage and concentration on the outcome of the YES. Compounds tested included new and established agonists, antagonists and negative controls, and results were evaluated according to prefixed statistical criteria. In addition, we assessed the assay’s performance in a blind interlaboratory validation exercise (IVE). An incubation time of five days was necessary to positively identify the estrogenic properties of all agonists tested, when dissolved in DMSO. Longer incubation times were required when using an ethanol protocol. Similar estrogenic activity was reported for benzyl butyl phthalate, bisphenol-A, methoxychlor, permethrin and genistein in the IVE. One out of the three laboratories did not classify α,β-endosulfan, dissolved in DMSO, as an estrogen. The same was true for 4,4′-DDE and lindane, dissolved in ethanol, a result that might be attributable to an inappropriate yeast start concentration and/or growth stage. These validation experiments show that under appropriate experimental conditions the YES yields sensitive, specific and reliable results. Therefore it fulfills the requirements as a first step screening assay to evaluate the capacity of chemicals to interact with the estrogen receptor.

Keywords: Yeast estrogen screen; Xenoestrogens; Sensitivity; Specificity; Interlaboratory validation

Analysis of hormonal steroids in fish plasma and bile by coupling solid-phase extraction to GC/MS by H. Budzinski; M. H. Devier; P. Labadie; A. Togola (pp. 1429-1439).

An analytical procedure for the simultaneous determination of twelve endogenous steroids (testosterone, androstenedione, 17β-estradiol, estrone, pregnenolone, progesterone, dihydroandrostenedione, dihydrotestosterone, 11α-ketotestosterone, 17α-hydroxyprogesterone, 17α-hydroxypregnenolone, 17α,20β-dihydroxy-4-pregnen-3-one) in plasma and bile samples by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC–MS) has been developed. After enzymatic hydrolysis for bile samples only, samples were concentrated and purified using two successive SPE (C₁₈ and NH₂) cartridges. Analytes were derivatized with a mixture of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) / mercaptoethanol / ammonium iodide (NH₄I) and determined by GC–MS in selective ion monitoring mode. For most of the steroids monitored, recoveries were in the range 90–120% in plasma and in the range 60–70% in bile, and the reproducibility was below 10% for the complete procedure. Limits of detection obtained ranged from 0.1 to 0.4 ng/g in fish plasma and from 1.6 to 14 ng/g in fish bile. The developed method was successfully applied to the determination of plasma steroids in flounders (Platichthys flesus) collected from two French estuaries.

Keywords: Steroids; Testosterone; Estradiol; Fish; Plasma; Bile; GC/MS

Immunoassay for acetamiprid detection: application to residue analysis and comparison with liquid chromatography by Eiki Watanabe; Shiro Miyake; Koji Baba; Heesoo Eun; Shozo Endo (pp. 1441-1448).

This work describes the fundamental ability of a commercial ELISA to determine acetamiprid and the application of the ELISA to residue analysis in fruit and vegetable samples. The ELISA exhibited satisfactory sensitivity (I ₅₀ 0.6 ng/g; limit of detection 0.053 ng/g) and a high selectivity for acetamiprid versus other neonicotinoid analogs (thiacloprid amide). Methanol, which influenced the sensitivity of the ELISA the least, was selected as the extractant for the ELISA analysis. Simple dilution of sample extracts with water eliminated matrix interferences. Average recoveries from the acetamiprid-spiked agricultural samples were >95% using a simple extraction method. Analytical results obtained from the ELISA were comparable to those obtained from the reference HPLC method (r>0.99). The ELISA applied to the residue analysis of acetamiprid in agricultural products is a rapid, simple, and cost-effective method, and could be successfully applied to the detection of acetamiprid before the distribution of produce.

Keywords: Acetamiprid; ELISA; Liquid chromatography; Pesticide residue analysis; Agricultural sample

Determination of frequently detected herbicides in water by solid-phase microextraction and gas chromatography coupled to ion-trap tandem mass spectrometry by François Perreau; Jacques Einhorn (pp. 1449-1456).

A method based on solid-phase microextraction (SPME) coupled with GC and ion trap tandem mass spectrometry has been developed for the analysis of nine herbicides and degradation products, among the most frequently found in natural water. A polydimethylsiloxane–divinylbenzene (PDMS–DVB)-coated fiber was selected to extract the analytes directly from the samples over the 0.01–1 μg L⁻¹ concentration range. Optimization of manual and automated SPME was performed on the basis of desorbed amounts, via various factorial experiment designs. Of the two modes, the automated one was found to be the most efficient. Memory effect was avoided owing to the 10-min fiber desorption time. Limits of detection reached down to below 0.01 μg L⁻¹ and repeatability ranged from 3 to 15% in natural water. A validation study was conducted involving the quantitation of the target compounds in Seine water with SPME/GC–MS-MS external calibration.

Keywords: Herbicide; Triazine; Degradation product; Solid-phase microextraction; GC–MS-MS analysis; Ion trap

Comparison of two solid-phase microextraction methods for the quantitative analysis of VOCs in indoor air by Virginie Larroque; Valérie Desauziers; Pierre Mocho (pp. 1457-1464).

Competitive adsorption on adsorptive solid-phase microextraction (SPME) fibres implies careful determination of operating conditions for reliable quantitative analysis of VOCs in indoor air. With this objective, two analytical approaches, involving non-equilibrium and equilibrium extraction, were compared. The average detection limit obtained for GC-MS analysis of nine VOCs by the equilibrium method is 0.2 μg m⁻³, compared with 1.9 μg m⁻³ with the non-equilibrium method. The effect of the relative humidity of the air on the calibration plots was studied, and shown to affect acetone adsorption only. Hence, the concentrations that can be accurately determined are up to 9 μmol m⁻³. The methods were then applied to indoor air containing different concentrations of VOCs. The non-equilibrium method, involving short extraction time, can be used for detection of pollution peaks whereas equilibrium extraction is preferable for measurement of sub-μg m⁻³ ground concentration levels.

Keywords: Solid-phase microextraction; PDMS-Carboxen; Indoor air; Volatile organic compounds; Humidity

A validated method for the determination of traces of UV filters in fish using LC–MS/MS by Maria Meinerling; Marion Daniels (pp. 1465-1473).

An analytical method for the determination of UV filter substances in fish tissue has been developed and validated using benzophenone-3, 3-(4-methylbenzylidene)-camphor, 2-ethylhexyl-2-cyano-3,3-diphenyl-2-propenoate and 2-ethylhexyl 3-(methoxyphenyl)-2-propenoate as target analytes. The fish fillets were homogenised and extracted by Soxhlet extraction. The extracts were run through a clean-up process including gel permeation chromatography followed by solid-phase extraction. Quantification of the compounds was performed using liquid chromatography with tandem mass spectrometric detection. Blank fish as well as spiked blank fish were analysed to validate the analytical method. The analytical method developed has the multiple advantages of enabling separation, simultaneous identification and quantification of each of the four selected compounds in a single run. Contamination of blank samples and abnormally high concentrations in spiked samples were avoided by taking extensive precautions during the fish preparation procedure. The method was validated in accordance with internationally accepted criteria, such as specificity, accuracy and repeatability. The combination of LC with tandem mass spectrometry ensures a high level of specificity. The accuracy of the method was reported as the mean recovery rate for the analytes in the sample matrix. Mean recoveries were in the range 86–108%. The precision is expressed as the relative standard deviation, and in all but one of the cases was 20% or below. The accuracy of the method allows residue analyses to be performed on biological matrices at ng/g levels. The determined limit of quantification for each analyte was 8 ng/g fish. For all spiking levels ≥8 ng/g, relative standard deviations were ≤ 20%.

Keywords: UV filter; LC–MS/MS; Traces in fish; Contamination

Determination of cadmium in oyster tissue using isotope dilution inductively coupled plasma mass spectrometry: comparison of results obtained in the standard and He/H₂ cell modes by Yiu-chung Yip; Hei-shing Chu; Kwok-chu Chan; Kam-kit Chan; Ping-yuk Cheung; Wing-cheong Sham (pp. 1475-1487).

An inductively coupled plasma quadrupole mass spectrometer equipped with an octopole collision/reaction cell was used for the determination of cadmium in oyster tissue samples using isotope dilution inductively coupled plasma mass spectrometry. The oyster samples in question were found to contain Mo and Zr. In our feasibility study on a Cd standard solution (10 μg L⁻¹) containing a matrix of Mo (1000 μg L⁻¹) or Zr (250 μg L⁻¹), the potentially interfering species (MoO⁺ or ZrO⁺) present at the analytical mass of cadmium concerned (m/z 111, 112 or 114) was reduced effectively through the use of a mixture of He and H₂ as cell gases. The accuracy of the method was validated by the analysis of a matrix-matched certified reference material (CRM) of NIST SRM 1566b. The CRM was analyzed under the standard and He/H₂ cell modes. Two isotopic pairs of ¹¹⁴Cd/¹¹¹Cd and ¹¹²Cd/¹¹¹Cd were selected for quantification purposes. The recoveries of cadmium obtained in the two cell modes were compared with each other. The validated method was applied successfully to the APMP.QM-P5 pilot study for international comparability purposes.

Keywords: Cadmium; Isotope dilution mass spectrometry; Collision/reaction cell; Oyster; APMP

Long-wavelength fluorimetry as an indirect detection system in immunoaffinity chromatography: application to environmental analysis by M. L. Sánchez-Martínez; M. P. Aguilar-Caballos; S. A. Eremin; A. Gómez-Hens (pp. 1489-1495).

The potential of long-wavelength fluorimetry when used as the detection system in immunoaffinity chromatography is assessed for the first time by applying this approach to the analysis of water and sludge samples. Nile blue (NB) was used to synthesize a long-wavelength fluorescent tracer for linear alkylbenzenesulfonates (LASs) using the carbodiimide method, in which the amino group of NB is covalently coupled to the activated carboxylic acid group of a LAS mimic with N-hydroxysuccinimide and dicyclohexylcarbodiimide. The method consists of the injection of a pre-incubated mixture containing linear sodium 4-dodecylbenzenesulfonate (LDS; used as the LAS model), anti-LAS antibodies, and the long-wavelength tracer into a commercial Protein G column. Free and bound tracer fractions are separated in the column, and the peak height of the immunochromatogram (corresponding to the free tracer) is directly measured at 626 nm (λ _ex) and 674 nm (λ _em), and then correlated to the analyte concentration. It is not necessary to perform an elution step immediately after every sample application. The dynamic range of the method is 0.05–2.5 μg ml⁻¹ LDS, and the detection limit is 15 ng ml⁻¹. The precision, expressed as the relative standard deviation, is 4.8–6.4%. Other surfactants (sodium dodecylsulfate and Triton X-100) do not cause interference. The recoveries obtained by applying the method to the analysis of water (ground- and wastewater) and sludge (primary and activated) samples ranged from 86.0 to 111.3%. Water sample analysis included an initial solid-phase extraction step, which cleaned up the samples and improved the detection limit fivefold.

Keywords: Immunoaffinity chromatography; Long-wavelength fluorimetry; LASs; Environmental samples

Improvement of HS-SPME for analysis of volatile organic compounds (VOC) in water samples by simultaneous direct fiber cooling and freezing of analyte solution by Elke Fries; Wilhelm Püttmann (pp. 1497-1503).

The sensitivity and precision of headspace solid-phase micro extraction (HS-SPME) at an analyte solution temperature (T _as) of +35 °C and a fiber temperature (T _fiber) of +5 °C were compared with those for HS-SPME at T _as and T _fiber of −20 °C for analysis of the volatile organic compounds benzene, 1,1,1-trichloroethane, trichloroethylene, toluene, o-xylene, ethylbenzene, m/p-xylene, and tetrachloroethylene in water samples. The effect of simultaneous fiber cooling and analyte solution freezing during extraction was studied. The compounds are of different hydrophobicity, with octanol/water partition coefficients (Kow) ranging from 126 and 2511. During a first set of experiments the polydimethylsiloxane (PDMS) SPME fiber was cooled to +5 °C with simultaneous heating of the aqueous analyte solution to +35 °C. During a second set of experiments, both SPME fiber holder and samples were placed in a deep freezer maintained at −20 °C for a total extraction time of 30 min. After approximately 2 min the analyte solution in the vial began to freeze from the side inwards and from the bottom upwards. After approximately 30 min the solution was completely frozen. Analysis of VOC was performed by coupling HS-SPME to gas chromatography-mass spectrometry (GC-MS). In general, i.e. except for tetrachloroethylene, the sensitivity of HS-SPME increased with increasing compound hydrophobicity at both analyte solution and fiber temperatures. At T _as of +35 °C and T _fiber of +5 °C detection limits of HS-SPME were 0.5 μg L⁻¹ for benzene, 1,1,1-trichloroethane, trichloroethylene, and tetrachloroethylene, 0.125 μg L⁻¹ for toluene, and 0.025 μg L⁻¹ for ethylbenzene, m/p-xylene, and o-xylene. In the experiments with T _as and T _fiber of −20 °C, detection limits were reduced for compounds of low hydrophobicity (Kow<501), for example benzene, toluene, 1,1,1-trichloroethane, and trichloroethylene. In the concentration range 0.5–62.5 μg L⁻¹, the sensitivity of HS-SPME was enhanced by a factor of approximately two for all compounds by performing the extraction at −20 °C. A possible explanation is that freezing of the water sample results in higher concentration of the target compounds in the residual liquid phase and gas phase (freezing-out), combined with enhanced adsorption of the compounds by the cooled fiber. The precision of HS-SPME, expressed as the relative standard deviation and the linearity of the regression lines, is increased for more hydrophobic compounds (Kow>501) by simultaneous direct fiber cooling and freezing of analyte solution. Background contamination during analysis is reduced significantly by avoiding the use of organic solvents.

Keywords: HS-SPME; VOC; Freezing-out; Volatile organic compounds; Water samples

Preparation, preservation and application of pure isotope-enriched phenyltin species by Dong Nguyen Van; Siva Rama Krishna Muppala; Wolfgang Frech; Solomon Tesfalidet (pp. 1505-1513).

A method combining liquid/liquid extraction and chromatographic fractionation has been developed for the preparation of pure monophenyltin (MPhT), diphenyltin (DPhT), and triphenyltin (TPhT), synthesized from isotope-enriched Sn metal using phenylation of SnI₄ in diethylether (DEE) followed by quenching with HBr and water. After two successive extractions of the aqueous HBr phase with DEE, >99% of both DPhT and TPhT was recovered in the combined DEE phase and 94% of the MPhT remained in the aqueous phase. The MPhT in the aqueous phase was extracted into dichloromethane. The organic phases were vaporized and the PhTs were redissolved in MeOH/water/acetic acid/sodium acetate (59/30/6/8, v/v/v/w), which was also used as storing solution. Aliquots of the two solutions containing either DPhT and TPhT or MPhT were injected into a silica-based C₁₈ column for isolating and purifying single species. The yields of pure MPhT, DPhT, and TPhT, each synthesized from isotope-enriched ¹¹⁸Sn metal, ¹²²Sn metal, and ¹²⁴Sn metal, were better than 99%. After chromatographic separation, the single phenyltin compounds were mixed to prepare a spike for multiple-isotope species-specific isotope dilution (MI-SSID). MI-SSID was successfully used to determine phenyltin compounds in the certified reference material, mussel tissue BCR CRM-477. At −20 °C, all of the fractionated phenyltin species were stable in the storage solution for at least 197 days. When these standards were stored at 4 °C or 22 °C, 4–6% of the DPhT and TPhT degraded within 27 days. The degradation of DPhT and TPhT increased with the ionic strength and acidity of the storage solution.

Keywords: Chromatographic fractionation; Phenyltin speciation; Multi-isotope species-specific isotope dilution; Production of phenyltin species; Inductively coupled plasma mass spectrometry

A rapid fractionation method for heavy metals in soil by continuous-flow sequential extraction assisted by focused microwaves by Tetsuya Nakazato; Mikio Akasaka; Hiroaki Tao (pp. 1515-1523).

A microwave-assisted continuous-flow sequential extraction system was developed for rapid fractionation analysis of heavy metals in soil. Insertion of pressure-adjusted air between the extractants provided stable flows of the extractants without mutual mixing and back-pressure influence of a column packed with soil, thereby facilitating reliable continuous-flow extractions. In addition, use of pure water as a pumping solvent removed metal contamination because of direct contact between corrosive extractants and the pump containing metallic materials. Focused microwave irradiation to the soil accelerated the selective extractions of the acid-soluble and reducible fractions of heavy metals in soil in the first and second steps of the sequential extraction conditions, as defined by the Commission of the European Bureau of Reference (BCR). The microwave-assisted continuous-flow extraction provided high correlations in amounts of six heavy metals except Zn in the first step and Cu in the second step extracted from a reference sludge soil, BCR CRM 483, with a conventional batchwise extraction proposed by BCR; continuous-flow extraction assisted by conductive heating provided lower correlations for all the six metals. The proposed method drastically reduced the time required for the sequence extraction to ca. 65 min without losing accuracy and precision of the fractionation analysis of heavy metals in soil, whereas the BCR batchwise method requires ca. 33 h.

Keywords: Metal; Soil; Extraction; Fractionation; Microwave; Continuous flow

Optimisation of pressurised liquid extraction for the determination of p,p′-DDT and p,p′-DDE in aged contaminated Ethiopian soils by Ahmed Hussen; Rikard Westbom; Negussie Megersa; Negussie Retta; Lennart Mathiasson; Erland Björklund (pp. 1525-1533).

The effectiveness of extracting p,p′-DDT and p,p′-DDE from aged contaminated soil samples by means of pressurized liquid extraction (PLE) was evaluated. Two soil samples, which were contaminated more than 10 years ago, were used in the investigation. The static extraction time was optimised and then validated against the total sum of target analytes obtained from multiple sequential extractions. The PLE results were also compared with Soxhlet extraction (SOX). PLE for 3×10 min at 100 °C was proven to be more exhaustive than SOX in the determination of p,p′-DDE from both soil samples. In the case of p,p′-DDT, PLE was found to be equally as exhaustive as SOX. Additionally, most of the previous PLE investigations used hazardous organic solvents such as n-hexane, toluene and dichloromethane mixed with acetone, whereas in this investigation the less toxic solvent combination n-heptane/acetone has been employed.

Keywords: Organochlorine pesticides; Endocrine disruptors; Pressurized liquid extraction; Soil; Trace organic compounds

Use of LIBS for rapid characterization of parchment by Bella Dolgin; Yuheng Chen; Valery Bulatov; Israel Schechter (pp. 1535-1541).

Parchment from different sources has been analyzed by laser-induced breakdown spectroscopy (LIBS) for determination of Ca, Na, K, Mg, Fe, Cu, and Mn. The LIBS results were compared with results from inductively coupled plasma spectroscopy (ICP) and good correlation was obtained. Rapid distinction between modern and historical samples was achieved by discriminant analysis of the LIBS data. Animal type recognition was also possible on the basis of Mg/Cu emission peak ratio and Mg depth profiling.

Keywords: Parchment; Identification; LIBS; Depth profile; ICP

Improved online coupling of planar chromatography with electrospray mass spectrometry: extraction of zones from glass plates by Alexander Alpmann; Gertrud Morlock (pp. 1543-1551).

A plunger-based extraction device for HPTLC/MS coupling, which was originally designed for extraction on TLC aluminum foils, was enhanced. The threefold modifications enabled extraction of analytes from glass-backed HPTLC/TLC plates after separation. A buffering of the plunger reduced the occurrence of leakage. The involvement of a torque screwdriver for the fixation resulted in a reproducible contact pressure and avoided breaking the glass plates. The employment of this device was also extended to plates with a layer thickness of 100 μm by reducing the height of the plunger’s cutting edge. Repeatability of the extraction from glass-backed plates, linearity of the signal obtained, and detection capability were shown to be comparable to the original device, which was only usable with aluminum foils. The influence of the elution solvent on the intensity of the MS signal was demonstrated to be a compromise between high elution power of the solvent and good solubility of the analyte in the elution solvent. The extraction device was employed for plates from different lots and for plates with different stationary phases thereby proving its general applicability in planar chromatography.

Keywords: HPTLC/TLC; Online extraction; HPTLC/ESI-MS; Electrospray ionization mass spectrometry

Near-infrared spectroscopy (NIRS) reflectance technology for the determination of tocopherols in alfalfa by I. González-Martín; J. M. Hernández-Hierro; M. Bustamante-Rangel; N. Barros-Ferreiro (pp. 1553-1558).

The vitamin E (α- and (β+γ)-tocopherol) contents present in alfalfa (fresh or dehydrated) were analysed using near-infrared spectroscopy (NIRS) technology together with a remote reflectance fibre-optic probe. The range of vitamin E was 0.55–5.16 mg/100 g for α-tocopherol and 0.07–0.48 for (β+γ)-tocopherol. The regression method employed was modified partial least squares (MPLS). The equations developed using the fibre-optic probe for 69 samples of alfalfa (dehydrated and fresh) to determine the content of vitamin E in feeds had multiple correlation coefficients (RSQs) and prediction corrected standard errors (SEP (C)) of 0.946 and 0.321 mg/100 g for α-tocopherol and 0.956 and 0.022 mg/100 g for (β+γ)-tocopherol. The predicted values of vitamin E in feeds using NIRS technology applying the fibre-optic probe directly on the sample with neither previous treatment nor manipulation are comparable to those obtained using the chemical method, which included alkaline hydrolysis and hexane extraction of the vitamin from the unsaponifiable fraction before chromatographic determination.

Keywords: Vitamin E; Tocopherols; Near-infrared spectroscopy; Fibre-optic probe; Alfalfa; Determination

Platinum electrode coated with a bentonite–carbon composite as an environmental sensor for detection of lead by Abdolkarim Abbaspour; Anahita Izadyar (pp. 1559-1565).

A Pt wire coated with a bentonite–carbon composite in a poly(vinyl chloride) membrane was used for detection of lead. The sensor has a Nernstian slope of 29.42±0.50 mV per decade over a wide range of concentration, 1.0×10⁻⁷ to 1.0×10⁻³ mol L⁻¹ Pb(NO₃)₂. The detection limit is 5.0×10⁻⁸ mol L⁻¹ Pb(NO₃)₂ and the electrode is applicable in the pH range 3.0–6.7. It has a response time of approximately 10 s and can be used at least for three months. The electrode has good selectivity relative to nineteen other metal ions. The practical analytical utility of the electrode is demonstrated by measurement of Pb(II) in industrial waste and river water samples.

Keywords: PVC-coated wire electrode; Carbon composite; Bentonite; Lead; Environment

Development of a bienzyme system for the electrochemical determination of nitrate in ambient air by Yue Cui; John P. Barford; Reinhard Renneberg (pp. 1567-1570).

This work reports the development of a bienzyme system consisting of salicylate hydroxylase (SHL) and nitrate reductase (NaR) for the electrochemical determination of nitrate. This method measures the concentration of nitrate directly under ambient air without suffering from oxygen interferences. The determination is based on the detection of NADH consumption, and the principle is as follows: NADH initiates the irreversible decarboxylation and hydroxylation of salicylate by SHL in the presence of oxygen to produce catechol, which results in a detectable signal due to its oxidation at the working electrode; the second enzyme, NaR, in the presence of nitrate, reduced the availability of NADH, and consequently, the current difference after the injection of nitrate is proportional to its concentration. This method shows high performance characteristics for nitrate determination with a broad detection range between 10 μM and 1,000 μM, a short measuring time of around 5 min, and a simple operation without sample pretreatment by inert gas purge or oxygen scavenger.

Keywords: Electrochemical determination; Nitrate; NADH; Nitrate reductase; Salicylate hydroxylase

Synchronous fluorescence spectroscopy for quantitative determination of virgin olive oil adulteration with sunflower oil by Konstantina I. Poulli; George A. Mousdis; Constantinos A. Georgiou (pp. 1571-1575).

Adulteration of extra virgin olive oil with sunflower oil is a major issue for the olive oil industry. In this paper, the potential of total synchronous fluorescence (TSyF) spectra to differentiate virgin olive oil from sunflower oil and synchronous fluorescence (SyF) spectra combined with multivariate analysis to assess the adulteration of virgin olive oil are demonstrated. TSyF spectra were acquired by varying the excitation wavelength in the region 270–720 nm and the wavelength interval (Δλ) in the region from 20 to 120 nm. TSyF contour plots for sunflower, in contrast to virgin olive oil, show a fluorescence region in the excitation wavelength range 325–385 nm. Fifteen different virgin olive oil samples were adulterated with sunflower oil at varying levels (0.5–95%) resulting in one hundred and thirty six mixtures. The partial least-squares regression model was used for quantification of the adulteration using wavelength intervals of 20 and 80 nm. This technique is useful for detection of sunflower oil in virgin olive oil at levels down to 3.4% (w/v) in just two and a half minutes using an 80-nm wavelength interval.

Keywords: Virgin olive oil; Total synchronous fluorescence spectroscopy; Adulteration; Partial least-squares (PLS); Food quality control

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Analytical and Bioanalytical Chemistry (v.386, #5)

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Analytical and Bioanalytical Chemistry (v.386, #5)