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Analytical and Bioanalytical Chemistry (v.387, #8)

Is your network robust? by John Fetzer (pp. 2601-2602).

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.

Is your network robust? by John Fetzer (pp. 2601-2602).

R.E. Wrolstad, T.E. Acree, E.A. Decker, M.H. Penner, D.S. Reid, S.J. Schwartz, C.F. Shoemaker, D. Smith, P. Sporns (Eds.): Handbook of food analytical chemistry – Water, proteins, enzymes, lipids, and carbohydrates by Franz Ulberth (pp. 2603-2604).

Randy D. Down and Jay H. Lehr (Eds.): Environmental instrumentation and analysis handbook by Bogusław Buszewski (pp. 2605-2606).

Lisa M. Balbes: Nontraditional careers for chemists. New formulas in chemistry by John C. Fetzer (pp. 2607-2608).

Fast and sensitive DNA analysis using changes in the FRET signals of molecular beacons in a PDMS microfluidic channel by Jaehyun Jung; Lingxin Chen; Sangyup Lee; Sungyong Kim; Gi Hun Seong; Jaebum Choo; Eun Kyu Lee; Chil-Hwan Oh; Sanghoon Lee (pp. 2609-2615).

A new DNA hybridization analytical method using a microfluidic channel and a molecular beacon-based probe (MB-probe) is described. A stem-loop DNA oligonucleotide labeled with two fluorophores at the 5′ and 3′ termini (a donor dye, TET, and an acceptor dye, TAMRA, respectively) was used to carry out a fast and sensitive DNA analysis. The MB-probe utilized the specificity and selectivity of the DNA hairpin-type probe DNA to detect a specific target DNA of interest. The quenching of the fluorescence resonance energy transfer (FRET) signal between the two fluorophores, caused by the sequence-specific hybridization of the MB-probe and the target DNA, was used to detect a DNA hybridization reaction in a poly(dimethylsiloxane) (PDMS) microfluidic channel. The azoospermia gene, DYS 209, was used as the target DNA to demonstrate the applicability of the method. A simple syringe pumping system was used for quick and accurate analysis. The laminar flow along the channel could be easily controlled by the 3-D channel structure and flow speed. By injecting the MB-probe and target DNA solutions into a zigzag-shaped PDMS microfluidic channel, it was possible to detect their sequence-specific hybridization. Surface-enhanced Raman spectroscopy (SERS) was also used to provide complementary evidence of the DNA hybridization. Our data show that this technique is a promising real-time detection method for label-free DNA targets in the solution phase. Figure FRET-based DNA hybridization detection using a molecular beacon in a zigzag-shaped PDMS microfluidic channel

Keywords: Fluorescence resonance energy transfer; Molecular beacon; Lab-on-a-chip; Surface-enhanced Raman; DNA hybridization

HPLC-ICP-MS and HPLC-ES-MS/MS characterization of synthetic seleno-arsenic compounds by Katerina Kanaki; Spiros A. Pergantis (pp. 2617-2622).

Various toxicological and metabolic interactions have been reported to exist between arsenic and selenium. In the present study, synthetic seleno-arsenic compounds, potentially suitable for probing metabolic interactions between these two elements, were prepared and tentatively characterized by using high-performance liquid chromatography (HPLC)–electrospray tandem mass spectrometry and HPLC–inductively coupled plasma mass spectrometry. In analogy to the recently identified thio-arsenic species, which can be prepared from their corresponding oxo-arsenic species via reaction with H₂S, the seleno-arsenic compounds were also derived from oxo-arsenic compounds via reaction with H₂Se. Figure H₂Se bubbled into solutions containing oxo‐arsenosugars converts them into their seleno‐arsenosugar analogues.

Keywords: Arsenic; Selenium; Speciation; Inductively coupled plasma mass spectrometry; Electrospray tandem mass spectrometry

HPLC-ICP-MS and HPLC-ES-MS/MS characterization of synthetic seleno-arsenic compounds by Katerina Kanaki; Spiros A. Pergantis (pp. 2617-2622).

Keywords: Arsenic; Selenium; Speciation; Inductively coupled plasma mass spectrometry; Electrospray tandem mass spectrometry

G-rich oligonucleotide-functionalized gold nanoparticle aggregation by Zai-Sheng Wu; Meng-Meng Guo; Guo-Li Shen; Ru-Qin Yu (pp. 2623-2626).

Guanine-rich DNA sequences commonly form helical quadruplex structures via Hoogsteen hydrogen bonds. The aggregation behavior of the nanoparticles, which are functionalized with four-guanine-terminated 27-base sequences at a nanoparticle-to-DNA ratio of 1:60, is investigated. To some extent, the guanine-quadruplex structures between the gold nanoparticles (GNPs) promote nanoparticle aggregation. However, the coordination site of the metal ion on the nanoparticle surface is partially passivated: the stability of guanine-rich DNA-GNPs is slightly lower than that of the usual DNA-GNPs, and the metal-ion specificity of nanoparticle assembly is substantially decreased. Thus, a mechanism for the aggregation of guanine-rich sequence-modified GNPs is proposed. It is possible to obtain a stable guanine-rich sequence-functionalized nanoparticle solution at high ionic strength by regulating guanine-rich DNA sequences. The controllability of guanine-rich sequence-modified nanoparticles makes the secondary structure of DNA a potentially useful candidate for DNA analysis and disease diagnostics. Figure Proposed mechanism for the aggregation of G-rich sequence-functionalized GNP

Keywords: Guanine-rich DNA sequences; Nanoparticle aggregation; G-quadruplex structure; UV-visible spectrum; Metal ion

G-rich oligonucleotide-functionalized gold nanoparticle aggregation by Zai-Sheng Wu; Meng-Meng Guo; Guo-Li Shen; Ru-Qin Yu (pp. 2623-2626).

Keywords: Guanine-rich DNA sequences; Nanoparticle aggregation; G-quadruplex structure; UV-visible spectrum; Metal ion

A novel microfluidic flow-injection analysis device with fluorescence detection for cation sensing. Application to potassium by Emilie Destandau; Jean-Pierre Lefèvre; Assia Chouai Fakhr Eddine; Serge Desportes; Marie Caroline Jullien; Rolland Hierle; Isabelle Leray; Bernard Valeur; Jacques Alexis Delaire (pp. 2627-2632).

A microfabricated device has been developed for fluorimetric detection of potassium ions without previous separation. It is based on use of a fluorescent molecular sensor, calix–bodipy, specially designed to be sensitive to and selective for the target ion. The device is essentially made of a Y-shape microchannel moulded in PDMS fixed on a glass substrate. A passive mixer is used for mixing the reactant and the analyte. The optical detection arrangement uses two optical fibres, one for excitation by a light-emitting diode, the other for collection of the fluorescence. This system enabled the flow-injection analysis of the concentration of potassium ions in aqueous solutions with a detection limit of 0.5 mmol L⁻¹ and without interference with sodium ions. A calibration plot was constructed using potassium standard solutions in the range 0–16 mmol L⁻¹, and was used for the determination of the potassium content of a pharmaceutical pill. Figure Photography of the microfluidic channel showing the ridges in the PDMS substrate at the top of the channel

Keywords: μTAS; Fluorimetric detection; Potassium sensor; Flow-injection analysis; Passive mixer

Determination of human erythropoietin by on-line immunoaffinity capillary electrophoresis: a preliminary report by Fernando Benavente; Elena Hernández; Norberto A. Guzman; Victoria Sanz-Nebot; José Barbosa (pp. 2633-2639).

Several CE methodologies have been described for the analysis of rHuEPO in concentrated solutions, but the inherently limited concentration sensitivity of CE precludes the detection of EPO at the levels found in biological fluids. In this work, we have investigated an on-line immunoaffinity solid-phase extraction capillary electrophoresis (IA-CE) methodology for the selective preconcentration of EPO in diluted solutions. The preliminary results obtained using a custom-made immunoaffinity sorbent prepared from an anti-human EPO polyclonal antibody and glutaraldehyde–glass beads show the potential of this novel approach. The summarized findings are discussed in detail as a starting point for our ongoing investigations.

Keywords: EPO; On-line preconcentration; Immunoaffinity; Solid-phase extraction; CE

Inhibitory analysis of the effect of polycyclic aromatic hydrocarbons on the activity of chitinase by means of liquid chromatography-mass spectrometry of chitin oligosaccharides by Yoriko Ooki; Momoko Kumemura; Masayoshi Itoh; Takashi Korenaga (pp. 2641-2644).

The analytical method of determining enzyme activity by liquid chromatography-mass spectrometry (LC/MS) was developed and applied for investigation of the effect of polycyclic aromatic hydrocarbons (PAHs) on the enzyme activity of chitinase. The measurement of chitinase activity by LC/MS is useful in order to use the nonderivatized substrate, which can show in vivo chitinase activity. Substrate consumption and product formation were monitored in order to determine chitinase activity. It was shown that, for the first time, in vitro addition of PAHs inhibited the activity of chitinase in a noncompetitive manner. The IC₅₀ value of benzo[a]pyrene was 1.4 μM, and PAHs containing four or more aromatic rings showed the same or higher inhibitory effect, whereas PAHs with a lower number of aromatic rings showed lower inhibition of the chitinase activity than benzo[a]pyrene.

Keywords: Chitinase; Polycyclic aromatic hydrocarbons; Inhibitor; Liquid chromatography-Mass spectrometry

Evaluation of amplified cRNA targets for oligonucleotide microarrays by Akihiro Sawada; Shogo Mizufune; Noritada Kaji; Manabu Tokeshi; Yoshinobu Baba (pp. 2645-2654).

Due to their hybridization specificity and capacity for systematic gene discovery, oligonucleotide-based microarray platforms offer numerous advantages over the cDNA microarrays currently widely used for comprehensive analysis of gene expression. Although fluorescently labeled amplified cRNA generated by T7 transcription is generally used in oligonucleotide microarrays, the feasibility of this combination (and that of cDNA microarrays) is yet to be studied systematically. In this paper, we performed a comparative study using a direct labeling method and T7 amplification to evaluate amplified cRNA targets for oligonucleotide microarrays. The efficiency of incorporation of Cy3- and Cy5-CTP into the target preparations, the reproducibility and the number of genes detected were investigated for each labeling approach and compared. The 12 genes that showed different expression profiles in the two labeling methods were evaluated by quantitative real-time PCR. In the 60-mer oligonucleotide microarray, amplified cRNA targets prepared by the T7 amplification method showed higher reproducibility and reliability than targets prepared by the direct labeling method in a comparative analysis of gene expression. This result also suggests the importance of fragmenting cRNA down to lengths of 50–200 bases before the hybridization process.

Keywords: 60-mer oligonucleotide microarray; Apoptosis; T7-based linear amplification; Quantitative real-time RT-PCR

Evaluation of amplified cRNA targets for oligonucleotide microarrays by Akihiro Sawada; Shogo Mizufune; Noritada Kaji; Manabu Tokeshi; Yoshinobu Baba (pp. 2645-2654).

Keywords: 60-mer oligonucleotide microarray; Apoptosis; T7-based linear amplification; Quantitative real-time RT-PCR

Towards rapid nanoscale chemical analysis using tip-enhanced Raman spectroscopy with Ag-coated dielectric tips by Boon-Siang Yeo; Thomas Schmid; Weihua Zhang; Renato Zenobi (pp. 2655-2662).

The influence of dielectric substrates on the Raman scattering activities of Ag overlayers has been investigated. Materials with low refractive indices, such as SiO₂, SiO_x and AlF₃, were found to provide suitable supporting platforms for Ag films to give strong surface-enhanced Raman scattering for dye molecules when illuminated at 488 nm. This finding was then extended to tip-enhanced Raman scattering (TERS). Huge enhancements of 70–80×, corresponding to net enhancements of >10⁴, were observed for brilliant cresyl blue test analyte when Ag-coated tips made from or precoated with low refractive index materials were applied. The yield of fabricated tips that significantly enhance the Raman signals was found to be close to 100%. These findings provide crucial steps towards the use of TERS as a robust technique for rapid chemical imaging with nanometer spatial resolution. Figure Silver-coated dielectric tips for tip-enhanced Raman scattering (TERS) are capable of more than 10,000-fold enhancement

Keywords: Nanoscale chemical analysis; Surface-enhanced Raman spectroscopy; Tip-enhanced Raman spectroscopy; Refractive index; Surface plasmon resonance

Towards rapid nanoscale chemical analysis using tip-enhanced Raman spectroscopy with Ag-coated dielectric tips by Boon-Siang Yeo; Thomas Schmid; Weihua Zhang; Renato Zenobi (pp. 2655-2662).

Keywords: Nanoscale chemical analysis; Surface-enhanced Raman spectroscopy; Tip-enhanced Raman spectroscopy; Refractive index; Surface plasmon resonance

Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy by Seong Ho Kang; Yun-Jeong Kim; Edward S. Yeung (pp. 2663-2671).

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM–aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.

Keywords: Single DNA molecules; Hybridization; Nanoarray chip; Dual color; Total internal reflection fluorescence microscopy (TIRFM)

Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy by Seong Ho Kang; Yun-Jeong Kim; Edward S. Yeung (pp. 2663-2671).

Keywords: Single DNA molecules; Hybridization; Nanoarray chip; Dual color; Total internal reflection fluorescence microscopy (TIRFM)

Drug profiling using planar microelectrode arrays by C. K. Yeung; F. Sommerhage; G. Wrobel; A. Offenhäusser; M. Chan; S. Ingebrandt (pp. 2673-2680).

Microelectrode arrays (MEAs) with evenly distributed multiple sensor spots have been designed for specific applications. Using the MEAs, we determined the relative profiles of potassium channel openers (KCOs) on cultured embryonic Sprague-Dawley rat cardiac myocytes. KCO, pinacidil (PIN), cromakalim (CROM), SDZ PCO400 (SDZ), or its vehicle, was added to the myocytes cumulatively. The action potential signal shapes in the presence of PIN and SDZ show that the changes in voltage over time and the magnitudes of the associated voltage change were reduced concentration-dependently. CROM affected sodium influx more than PIN and SDZ. The comparisons of changes in the rate of beating and propagation speed in the presence of KCOs were made using their corresponding pD₂ values (the negative log of EC₅₀). All KCOs caused concentration-dependent reductions in the rate of beating and propagation speed, with SDZ being the most potent. In addition to the signal shapes, rate of beating, and propagation speed, the origin of excitation and the excitation pattern inside the culture can be also extracted. The results show that the present system can differentiate the effects of different KCOs on myocytes. It might be possible to utilise the MEA as a means to classify drug action based upon a combined interpretation of the three different datasets gained from the extracellular recordings. The combination of these observations might be used as ‘drug signatures’ when profiling drugs in the future.

Keywords: Microelectrode array; Potassium channel openers; Pharmacology; Bioassay; Cardiac myocytes

Drug profiling using planar microelectrode arrays by C. K. Yeung; F. Sommerhage; G. Wrobel; A. Offenhäusser; M. Chan; S. Ingebrandt (pp. 2673-2680).

Keywords: Microelectrode array; Potassium channel openers; Pharmacology; Bioassay; Cardiac myocytes

Preparation and evaluation of a new synthetic polymeric chiral stationary phase for HPLC based on the trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid bis-4-vinylphenylamide monomer by Xinxin Han; Chunlei Wang; Lingfeng He; Thomas E. Beesley; Daniel W. Armstrong (pp. 2681-2697).

A new synthetic polymeric chiral stationary phase for liquid chromatography was prepared via free-radical-initiated polymerization of trans-9,10-dihydro-9,10-ethanoanthracene-(11S,12S)-11,12-dicarboxylic acid bis-4-vinylphenylamide. The new polymeric chiral stationary phase (CSP) showed enantioselectivity for many chiral compounds in multiple mobile phases. High stability and sample capacities were observed on this polymeric chiral stationary phase. Mobile phase components and additives affected chiral separation greatly. This new synthetic chiral stationary phase is complementary to two other related commercially available CSPs: the P-CAP and P-CAP-DP columns. Interactions between the chiral stationary phase and analytes that lead to retention and chiral recognition include hydrogen bonding, dipolar, and π–π interactions. Repulsive (steric) interactions also contribute to chiral recognition. Figure LC chromatograms showing the analytical (blue) and preparative (red) separations of N-(3,5-dinitrobenzoylleucine) enantiomers on a new synthetic polymeric chiral stationary phase

Keywords: Chiral stationary phase (CSP); Enantioselectivity; Polymeric CSP; Preparative chromatographic separation; Normal phase LC

Polycation coating poly(dimethylsiloxane) capillary electrophoresis microchip for rapid separation of ascorbic acid and uric acid by Q. L. Zhang; J. J. Xu; H. Z. Lian; X. Y. Li; H. Y. Chen (pp. 2699-2704).

A novel method for rapid separation and determination of ascorbic acid and uric acid has been developed with a polycation-modified poly(dimethylsiloxane) (PDMS) microchip under a negative-separation electric field. Just by flushing the microchip with aqueous solutions of the polycations, poly(allylamine) hydrochloride, poly(diallyldimethylammonium chloride) or chitosan could be stably coated on the PDMS microchannel surface, which resulted in a reversed electroosmotic flow and thus the rapid and efficient separation of the two substrates. Factors influencing the separation, including polycation category, buffer solution, detection potential and separation voltage, were investigated and optimized. The cheapness, rapid analysis speed and the successful analysis of human urine make this microsystem attractive for application in clinics. Figure The electropherograms of 100 μ/mL AA and UA in (1) PAH, (2) PDDA, (3) Chitosan modified PDMS microchannels and native PDMS microchip (4).

Keywords: Poly(dimethylsiloxane) microchip; Polyelectrolyte coating; Amperometry; Ascorbic acid; Uric acid

Study of a new derivatizing reagent that improves the analysis of amino acids by HPLC with fluorescence detection: application to hydrolyzed rape bee pollen by Jinmao You; Lingjun Liu; Wenchen Zhao; Xianen Zhao; Yourui Suo; Honglun Wang; Yulin Li (pp. 2705-2718).

A simple and sensitive method for evaluating the chemical compositions of protein amino acids, including cystine (Cys)₂ and tryptophane (Try) has been developed, based on the use of a sensitive labeling reagent 2-(11H-benzo[α]-carbazol-11-yl) ethyl chloroformate (BCEC–Cl) along with fluorescence detection. The chromophore of the 1,2-benzo-3,4-dihydrocarbazole-ethyl chloroformate (BCEOC-Cl) molecule was replaced with the 2-(11H-benzo[α]-carbazol-11-yl) ethyl functional group, yielding the sensitive fluorescence molecule BCEC–Cl. The new reagent BCEC–Cl could then be substituted for labeling reagents commonly used in amino acid derivatization. The BCEC–amino acid derivatives exhibited very high detection sensitivities, particularly in the cases of (Cys)₂ and Try, which cannot be determined using traditional labeling reagents such as 9-fluorenyl methylchloroformate (FMOC-Cl) and ortho-phthaldialdehyde (OPA). The fluorescence detection intensities for the BCEC derivatives were compared to those obtained when using FMOC-Cl and BCEOC-Cl as labeling reagents. The ratios I _BCEC/I _BCEOC = 1.17–3.57, I _BCEC/I _FMOC = 1.13–8.21, and UV_BCEC/UV_BCEOC = 1.67–4.90 (where I is the fluorescence intensity and UV is the ultraviolet absorbance). Derivative separation was optimized on a Hypersil BDS C₁₈ column. The detection limits calculated from 1.0 pmol injections, at a signal-to-noise ratio of 3, ranged from 7.2 fmol for Try to 8.4 fmol for (Cys)₂. Excellent linear responses were observed, with coefficients of >0.9994. When coupled with high-performance liquid chromatography, the method established here allowed the development of a highly sensitive and specific method for the quantitative analysis of trace levels of amino acids including (Cys)₂ and Try from bee-collected pollen (bee pollen) samples.

Keywords: Rape bee pollen; Amino acids; 2-(11H-Benzo[α]-carbazol-11-yl) ethyl chloroformate (BCEC–Cl)

Stacking and quantitative analysis of lovastatin in urine samples by the transient moving chemical reaction boundary method in capillary electrophoresis by Min Li; Liu-Yin Fan; Wei Zhang; Cheng-Xi Cao (pp. 2719-2725).

A simple, sensitive, and useful concentration method for lovastatin (Lvt) in urine has been developed based on the transient moving chemical reaction boundary method (tMCRBM) in capillary electrophoresis. The MCRB is formed with acidic sample buffer (Gly-HCl) and alkaline running buffer (Gly-NaOH). The following optimal conditions were determined for stacking and separation: electrophoretic buffer of 100 mM Gly- NaOH (pH 11.52), sample buffer of 20 mM Gly-HCl (pH 4.93), fused-silica capillary of 76 cm × 75-μm i.d (67 cm from detector), sample injection at 14 mbar for 3 min. A 21- to 26-fold increase in peak height was achieved for detection of Lvt in urine under the optimal conditions compared with normal capillary zone electrophoresis. By combining the sample pretreatment procedure with the stacking method, the sensitivity of Lvt in urine was increased by 105- to 130-fold. The limits of detection (LOD) and quantification (LOQ) for Lvt in urine were decreased to 8.8 ng/mL and 29.2 ng/mL, respectively. The intra-day and inter-day precision values (expressed as RSD) were 2.23–3.61% and 4.03–5.05%, respectively. The recoveries of the analyte at three concentration levels changed from 82.65 to 100.49%.

Keywords: Capillary zone electrophoresis; Stacking; Lovastatin; Moving chemical reaction boundary; Urine

Enhanced sensitivity of self-assembled-monolayer-based SPR immunosensor for detection of benzaldehyde using a single-step multi-sandwich immunoassay by K. Vengatajalabathy Gobi; Kiyoshi Matsumoto; Kiyoshi Toko; Hidekazu Ikezaki; Norio Miura (pp. 2727-2735).

This paper describes the fabrication and sensing characteristics of a self-assembled monolayer (SAM)-based surface plasmon resonance (SPR) immunosensor for detection of benzaldehyde (BZ). The functional sensing surface was fabricated by the immobilization of a benzaldehyde–ovalbumin conjugate (BZ–OVA) on Au-thiolate SAMs containing carboxyl end groups. Covalent binding of BZ–OVA on SAM was found to be dependent on the composition of the base SAM, and it is improved very much with the use of a mixed monolayer strategy. Based on SPR angle measurements, the functional sensor surface is established as a compact monolayer of BZ–OVA bound on the mixed SAM. The BZ–OVA-bound sensor surface undergoes immunoaffinity binding with anti-benzaldehyde antibody (BZ-Ab) selectively. An indirect inhibition immunoassay principle has been applied, in which analyte benzaldehyde solution was incubated with an optimal concentration of BZ-Ab for 5 min and injected over the sensor chip. Analyte benzaldehyde undergoes immunoreaction with BZ-Ab and makes it inactive for binding to BZ–OVA on the sensor chip. As a result, the SPR angle response decreases with an increase in the concentration of benzaldehyde. The fabricated immunosensor demonstrates a low detection limit (LDL) of 50 ppt (pg mL⁻¹) with a response time of 5 min. Antibodies bound to the sensor chip during an immunoassay could be detached by a brief exposure to acidic pepsin. With this surface regeneration, reusability of the same sensor chip for as many as 30 determination cycles has been established. Sensitivity has been enhanced further with the application of an additional single-step multi-sandwich immunoassay step, in which the BZ-Ab bound to the sensor chip was treated with a mixture of biotin-labeled secondary antibody, streptavidin and biotin–bovine serum albumin (Bio–BSA) conjugate. With this approach, the SPR sensor signal increased by ca. 12 times and the low detection limit improved to 5 ppt with a total response time of no more than ca. 10 min. Figure A single-step multi-sandwich immunoassay step increases SPR sensor signal by ca. 12 times affording a low detection limit for benzaldehyde of 5 ppt

Keywords: Surface plasmon resonance; Immunosensor; Fragrance sensor; Low molecular weight analyte; Food-related sensor application; Self-assembled monolayer

New FRET primers for quantitative real-time PCR by Ashraf I. Ahmad; Jahan B. Ghasemi (pp. 2737-2743).

FRET primer real-time PCR chemistry depends on internally labeled primers with FRET dyes linked to their 3′ end. The best distance between the FRET dyes for obtaining the largest signal and the lowest background is six nucleotides. In this study the forward primer was labeled with FAM and the reverse primer with Texas red; the labeled primers meet in cycle two of PCR. At the end of the elongation step FAM is excited to emit fluorescence which will excite Texas red to emit new fluorescence that correlates directly with the quantity of PCR product accumulated. FRET primer techniques amplify short amplicons with unique thermal cycling steps, 0 s at 85 °C for denaturation, 7 s for annealing, and 2 s for elongation. The FRET primer technique was very efficient (92.6, 97.2, and 100%), correlation coefficients were high (1.0, 0.999, and 0.999), and total run time was very short (20, 45, and 40 min per 40 cycles with LightCycler, iCycler, and RotorGene 3000, respectively). When FRET-labeled primers were compared with similar but unlabeled primers it was observed that the FRET primer technique had a lower Ct value and was more efficient than use of unlabeled primers detected by use of SYBR Green I. Figure Schematic diagram of FRET prime real-time PCR

Keywords: Real-time PCR; FRET Primers; Labeled primers; Quantitative real-time PCR; FAM; Texas red

New FRET primers for quantitative real-time PCR by Ashraf I. Ahmad; Jahan B. Ghasemi (pp. 2737-2743).

Keywords: Real-time PCR; FRET Primers; Labeled primers; Quantitative real-time PCR; FAM; Texas red

Accurate determination of an immunosuppressant in stented swine tissues with LC–MS/MS by Jun Zhang; Micheal T. Reimer; Qin C. Ji; Min S. Chang; Tawakol A. El-Shourbagy; Sandra Burke; Lewis Schwartz (pp. 2745-2756).

During stent development, accurate monitoring of the drug concentration in animal tissues can provide critical information on how the drug is released into the circulation and the surrounding tissues. To establish the relationship between the drug concentration and the distance from the stent to the target tissue, a comprehensive strategy was developed for sample collection, sample homogenization and sample storage as well as sample analysis. This strategy was developed with the analytical chemists and animal surgical specialists working together as a team. The optimized sampling process was designed to yield a representative sample, appropriately located and of an appropriate size. The sampling process was also designed to eliminate the potential for carryover and cross-contamination. During sample processing, the analyte solution was spiked into blank tissues using a sharp needle and a gas-tight syringe to prepare tissue quality control samples. These tissue quality controls were then used to evaluate the stability of the drug in solid tissue and homogenate, the homogenization carryover, the cross-contamination and the recovery of the drug during method validation and to monitor the overall process of drug analysis of the swine tissues. This thorough strategy has been applied to the accurate determination of zotarolimus in swine tissues for regulated toxicology studies. The entire process was controlled, including precise tissue sampling, compound-based tissue homogenization, method validation, and the application of the method to regulated toxicokinetics studies. The results demonstrate that analytical chemistry concepts can be successfully integrated into toxicokinetics studies in order to collect precise samples and obtain meaningful results. The strategy can be applied to similar toxicokinetics studies of locally administrated drugs in tissues.

Keywords: Zotarolimus; Tissue; Sampling; Homogenization; LC–MS/MS

On-line determination of 3,5,6-trichloro-2-pyridinol in human urine samples by surface plasmon resonance immunosensing by E. Mauriz; A. Calle; J. J. Manclús; A. Montoya; L. M. Lechuga (pp. 2757-2765).

An immunochemical method for the analysis of 3,5,6-trichloro-2-pyridinol (TCP), a major urinary metabolite of chlorpyrifos, is developed using a surface plasmon resonance (SPR)-based biosensor. The stability of the assay was assessed by covalently linking the analyte derivative to a thin, gold-modified sensor surface. For optimization of analyte derivative immobilization, sensor chips were activated via alkanethiol monolayers with terminal amine or carboxyl groups. Binding inhibition tests were performed in untreated urine samples and compared to those obtained in distilled water and PBS was used as control. In all cases, similar detection limits, at the micrograms per litre level (0.1–0.24 μg L⁻¹), were attained for TCP assays independently of the dilution buffer. Reproducibility of measurements was studied throughout more than 130 regeneration cycles, which allowed the repeated use of the same immunosensor surface without significant variation of the SPR signal. All measurements were developed in real-time in only 10 min, using a SPR portable system. The device could be applied as a valuable analytical method to both environmental screening and clinic diagnostics.

Keywords: TCP; Chlorpyrifos; SPR immunosensor; On-line determination; Urine biomarkers; Clinic diagnostics

Simultaneous use of multiple fluorescent reporter dyes for glucose sensing in aqueous solution by David B. Cordes; Aaron Miller; Soya Gamsey; Bakthan Singaram (pp. 2767-2773).

The simultaneous use of several fluorescent reporter dyes in a multicomponent boronic acid-based glucose sensing system is reported. In one application, two dyes with widely different emission wavelengths are used to report changes in glucose concentration. A third glucose-insensitive dye was then added to act as a reference dye and provide for a ratiometric correction to the two reporter dye signals. The inclusion of such a reference dye reduces errors arising from sources such as fluctuations in lamp intensity and sample dilution. The simultaneous use of multiple fluorescent reporter dyes

Keywords: Carbohydrates; Glucose sensing; Boronic acid; Fluorescence; Viologen

Simultaneous use of multiple fluorescent reporter dyes for glucose sensing in aqueous solution by David B. Cordes; Aaron Miller; Soya Gamsey; Bakthan Singaram (pp. 2767-2773).

Keywords: Carbohydrates; Glucose sensing; Boronic acid; Fluorescence; Viologen

Fluorescent determination of cardiolipin using 10-N-nonyl acridine orange by P. Kaewsuya; N. D. Danielson; D. Ekhterae (pp. 2775-2782).

Cardiolipin (CL) plays an essential role as a marker for cell apoptosis. Quantitative detection of phospholipids (PLs) by UV absorbance is problematic due to the presence of few double bonds in the structure. Although 10-N-nonyl acridine orange (NAO) has been utilized for fluorescent visualization of liposomes and mitochondria through its interaction with CL, in this work, we have developed a specific fluorescent method for CL in solution using NAO. The interaction of sodium n-dodecyl sulfate (SDS), used to treat cells prior to lipid extraction, and other PLs found in cell membranes such as phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidiylserine (PS), and sphingomyelin (SM) with NAO is investigated. The fluorescence intensity of the 0.5 μM NAO signal is strongly quenched by SDS below 25% methanol in water but with a methanol content above 50%, no quenching of NAO by SDS is observed. No fluorescence quenching of NAO with a 50% methanol/50% water solvent by the previously mentioned PLs or 4–20 μM cholesterol with the exception of PG at above 8 μM is noted. Using this 50% methanol/50% water solvent, the fluorescence signal due to the NAO–CL interaction is quite stable from 3 to at least 15 min. With excitation and emission wavelengths set at 518 and 530 nm, respectively, 20 μM NAO provides an inverse linear fluorescence response at 0.2–10 μM CL with a correlation coefficient of 0.9929. The detection limit is 0.2 μM and the limit of quantification is 0.6 μM. Structurally analogous acridine orange and phenosafranin dyes are less effective as fluorescent probes for CL. The CL in the whole cell and membrane samples is quantitatively determined by standard addition to range from 0.2 to 1.5 μM. The level of CL in cell membrane samples, previously subjected to staurosporine which initiates cell apoptosis, is increased but not significantly through use of the t-test.

Keywords: Fluorescence; Cardiolipin; Phospholipids; 10-N-nonyl acridine orange

Fluorescent determination of cardiolipin using 10-N-nonyl acridine orange by P. Kaewsuya; N. D. Danielson; D. Ekhterae (pp. 2775-2782).

Keywords: Fluorescence; Cardiolipin; Phospholipids; 10-N-nonyl acridine orange

Simultaneous determination of the advanced glycation end product N ^ɛ-carboxymethyllysine and its precursor, lysine, in exhaled breath condensate using isotope-dilution–hydrophilic-interaction liquid chromatography coupled to tandem mass spectrometry by T. Schettgen; A. Tings; C. Brodowsky; A. Müller-Lux; A. Musiol; T. Kraus (pp. 2783-2791).

Analysis of biomarkers in exhaled breath condensate (EBC) is a non-invasive method for investigating the effects of different diseases or exposures, on the lungs and airways. N ^ɛ-carboxymethyllysine (CML) is an important biomarker of advanced glycation end products (AGEs). A method has been developed for simultaneous determination of CML and its precursor, the amino acid lysine, in exhaled breath condensate (EBC). After addition of labelled internal standards (d-4-CML; d-4-lysine), the EBC was concentrated by freeze-drying. Separation and detection of the analytes were performed by hydrophilic-ion liquid chromatography coupled with tandem mass-spectrometric detection (HILIC–MS–MS). The limits of quantification were 10 pg mL⁻¹ EBC and 0.5 ng mL⁻¹ EBC for CML and lysine, respectively. The relative standard deviation of the within-series precision was between 2.8 and 7.8% at spiked concentrations between 40 and 200 pg mL⁻¹ for CML and between 6 and 20 ng mL⁻¹ for lysine. Accuracy for the analytes ranged between 89.5 and 133%. The method was used for the analysis of EBC samples from ten healthy persons from the general population and ten persons receiving dialysis. CML and lysine were detected in all EBC samples with median values of 19 pg mL⁻¹ CML and 11.9 ng mL⁻¹ lysine in EBC of healthy persons and 25 pg mL⁻¹ CML and 9.5 ng mL⁻¹ lysine in EBC of dialysis patients.

Keywords: Exhaled breath condensate; Advanced glycation end product; HILIC; CML

A simple electroanalytical methodology for the simultaneous determination of dopamine, serotonin and ascorbic acid using an unmodified edge plane pyrolytic graphite electrode by Roohollah Torabi Kachoosangi; Richard G. Compton (pp. 2793-2800).

A simple method using an unmodified edge plane pyrolytic graphite electrode (EPPGE) is reported for the simultaneous determination of dopamine (DA), serotonin (ST) and ascorbic acid (AA). The performance of this electrode is superior to other unmodified carbon-based electrodes and also to many modified electrodes in terms of detection limit, sensitivity and peak separation for determination of DA, ST and AA. Using this method, detection limits of 90 nM, 60 nM and 200 nM were obtained for DA, ST and AA respectively. No electrode fouling is observed during a set of experiments and good sensitivity is obtained for the simultaneous determination of DA, ST and AA. The peaks for the three species are well resolved from each other and the electrode is successfully utilised for their determination in standard and real samples.

Keywords: EPPGE; Dopamine; Serotonin; Ascorbic acid; Simultaneous determination

Hydroxymethylfurfural: an enemy or a friendly xenobiotic? A bioanalytical approach by K. Michail; V. Matzi; A. Maier; R. Herwig; J. Greilberger; H. Juan; O. Kunert; R. Wintersteiger (pp. 2801-2814).

Hydroxymethylfurfural (HMF), a well-known heterocyclic Maillard reaction product, has often been studied for its potential toxic, mutagenic, and carcinogenic effects. Recent clinical studies, however, have strongly suggested that HMF might have exciting antitumor potential. We report on the development and validation of a bioanalytical assay for HMF that could be suitable as a basis for pharmacokinetic models in cancer patients. Two strategies were tested, i.e., direct and indirect methodologies. A direct isocratic LC determination at 283 nm was designed. Two indirect attempts involved derivatization coupled to HPLC-UV. It was possible to resolve the stereoisomers of the HMF derivative, and factors influencing their equilibrium ratio are discussed. HMF was extracted from the biomatrix by solid-phase extraction using different cartridges. A comparative study was made of the implemented methods as well as the extraction protocols. Both indirect assays proved to be more sensitive and were used to assess HMF quantitatively in human plasma. However, the newly introduced derivatization conditions led to the highest sensitivity with a LOD (S/N ratio = 3) of at least 2 pmol analyte on column. The assay selectivity was satisfactory in pre- and post-dose real samples. The mean recoveries of the assays were 79% and 89%, with acceptable accuracies and reproducibilities. Figure Schematic representation of hydroxymethylfurfural (HMF) in human plasma

Keywords: Hydroxymethylfurfural; Derivatization; HPLC-UV; Validation; Human plasma

A study of the interactions between carboplatin and blood plasma proteins using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry by Ruimin Xie; Willie Johnson; Lorna Rodriguez; Murugesan Gounder; Gene S. Hall; Brian Buckley (pp. 2815-2822).

To study the carboplatin–protein interaction, a sensitive method using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC–ICP–MS) was developed. The complexes formed between plasma proteins and carboplatin were monitored and identified with this method. Composite blood plasma samples from patients who were undergoing chemotherapy were analyzed, and carboplatin was found to bind plasma proteins. In addition, blank plasma samples were spiked with carboplatin and were analyzed as a time course study, and the results confirmed that carboplatin formed complexes with plasma proteins, primarily albumin and γ-globulin. To further substantiate the study, these two proteins were incubated with carboplatin. The binding between carboplatin and these proteins was then characterized qualitatively and quantitatively. In addition to a one-to-one binding of Pt to protein, protein aggregation was observed. The kinetics of the binding process of carboplatin to albumin and γ-globulin was also studied. The initial reaction rate constant of carboplatin binding to albumin was determined to be 0.74 M⁻¹ min⁻¹, while that for γ-globulin was 1.01 M⁻¹ min⁻¹, which are both lower than the rate constant of the cisplatin–albumin reaction previously reported.

Keywords: Metal speciation; Carboplatin; Protein complexation; SEC–ICP–MS

Chemotaxonomical identification of spores of macrofungi: possibilities of Raman spectroscopy by Kris De Gussem; Peter Vandenabeele; Annemieke Verbeken; Luc Moens (pp. 2823-2832).

Confocal Raman spectroscopy is a non-destructive analytical method which is useful to obtain detailed information about the molecular composition of biological samples. Its high spatial resolution was used to collect spectra of single basidiospores of macrofungi of the genera Collybia, Gymnopus, Laccaria, Lactarius, Mycena and Russula. These spectra can be divided into three major taxon-related groups, with general compositional differences, such as the relative amount of lipids compared to proteins. In this study, collapsing of thin-walled spores during storage was often observed, a phenomenon which has been given little attention in the literature. The Raman spectra are treated with different chemometric preprocessing techniques, including Savitsky–Golay, standard normal variate (SNV) preprocessing and extended multiplicative scatter correction (EMSC). By using linear discriminant analysis, approximately 90% of the spectra can be assigned to the correct genus, but identification on the species level was not possible.

Keywords: Raman spectroscopy; Basidiomycotina; Chemotaxonomy; Genus identification

Chemotaxonomical identification of spores of macrofungi: possibilities of Raman spectroscopy by Kris De Gussem; Peter Vandenabeele; Annemieke Verbeken; Luc Moens (pp. 2823-2832).

Keywords: Raman spectroscopy; Basidiomycotina; Chemotaxonomy; Genus identification

Estimation of binding constants of receptors and ligands by affinity capillary electrophoresis by Li-Wei Zhang; Li Ding; Xin-Xiang Zhang (pp. 2833-2841).

An estimation method for determination of binding constants of receptors to ligands by affinity capillary electrophoresis was evaluated. On the basis of the theories of pseudostationary phase or so-called dynamic stationary phase, the retention factor (k) was used to represent the interaction between the receptor and ligand. k could be easily deduced from the migration times of the ligand and the receptor. Then, with the linear relationship of k versus the concentration of ligand in the running buffer, the binding constant K _b was calculated from the slope and intercept. In order to test its feasibility, the calculation method was demonstrated using three model systems: the interactions between vancomycin and N-acetyl-d-Ala-d-Ala, ristocetin and N-acetyl-d-Ala-d-Ala, and carbonic anhydrase B and an arylsulfonamide. Estimated binding constants were compared with those determined by other techniques. The results showed that this estimation method was reliable. This calculation method offers a simple and easy approach to estimating binding constants of ligands to receptors.

Keywords: Affinity capillary electrophoresis; Binding constant; Retention factor; Migration time

Estimation of binding constants of receptors and ligands by affinity capillary electrophoresis by Li-Wei Zhang; Li Ding; Xin-Xiang Zhang (pp. 2833-2841).

Keywords: Affinity capillary electrophoresis; Binding constant; Retention factor; Migration time

¹⁹F NMR spectroscopic characterization of the interaction of niflumic acid with human serum albumin by Keisuke Kitamura; Ahmed A. Omran; Shigehiko Takegami; Rumi Tanaka; Tatsuya Kitade (pp. 2843-2848).

The interaction of a non-steroidal anti-inflammatory drug, niflumic acid (NFA), with human serum albumin (HSA) has been investigated by ¹⁹F nuclear magnetic resonance (NMR) spectroscopy. A ¹⁹F NMR spectrum of NFA in a buffered (pH 7.4) solution of NaCl (0.1 mol L⁻¹) contained a single sharp signal of its CF₃ group 14.33 ppm from the internal reference 2,2,2-trifluoroethanol. Addition of 0.6 mmol L⁻¹ HSA to the NFA buffer solution caused splitting of the CF₃ signal into two broadened signals, shifted to the lower fields of 14.56 and 15.06 ppm, with an approximate intensity ratio of 1:3. Denaturation of HSA by addition of 3.0 mol L⁻¹ guanidine hydrochloride (GU) restored a single sharp signal of CF₃ at 14.38 ppm, indicating complete liberation of NFA from HSA as a result of its denaturation. These results suggest that the binding is reversible and occurs in at least two HSA regions. Competitive ¹⁹F NMR experiments using warfarin, dansyl-l-asparagine, and benzocaine (site I ligands), and l-tryptophan and ibuprofen (site II ligands) revealed that NFA binds to site I at two different regions, Ia and Ib, in the ratio 1:3. By use of ¹⁹F NMR with NFA as an ¹⁹F NMR probe the nonfluorinated site I-binding drugs sulfobromophthalein and iophenoxic acid were also found to bind sites Ia and Ib, respectively. These results illustrate the usefulness and convenience of ¹⁹F NMR for investigation of the HSA binding of both fluorinated and nonfluorinated drugs.

Keywords: Niflumic acid; ¹⁹F NMR spectroscopy; Human serum albumin; Binding; Site Ia; Site Ib

Electrothermal vaporization dynamic reaction cell inductively coupled plasma mass spectrometry for the determination of Fe, Co, Ni, Cu, and Zn in biological samples by Yen-Jia Tseng; Yu-Duan Tsai; Shiuh-Jen Jiang (pp. 2849-2855).

Slurry sampling electrothermal vaporization dynamic reaction cell inductively coupled plasma mass spectrometry (ETV-DRC-ICP-MS) has been applied to determine Fe, Co, Ni, Cu, and Zn in biological samples. The influences of instrument operating conditions and slurry preparation on the ion signals were reported. Pd was used as the modifier. The effectiveness of the ETV sample introduction technique and dynamic reaction cell in alleviating various spectral interferences in ICP-MS analysis has been demonstrated. This method has been applied to determine Fe, Co, Ni, Cu, and Zn in NIST SRM 1573a tomato leaves reference material and NRCC DORM-2 dogfish muscle reference material and also real samples such as a tea and a swordfish sample purchased locally. Since the sensitivities of the elements studied in slurry and aqueous solution were different, an analyte addition technique was used for the determinations. The analytical results of the reference materials agreed with the certified values. The precision between sample replicates was better than 6% for all determinations. The method detection limit estimated from analyte addition curves was 0.01, 0.006, 0.007, 0.004, and 0.006 μg g⁻¹ for Fe, Co, Ni, Cu, and Zn, respectively, in the original biological samples.

Keywords: Dynamic reaction cell inductively coupled plasma mass spectrometry; Slurry sampling; Electrothermal vaporization; Biological samples; Fe, Co, Ni, Cu, and Zn

Spectrophotometric determination of total protein in serum using a novel near-infrared cyanine dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine by Hong Wang; Wei-Rong Li; Xiao-Feng Guo; Hua-Shan Zhang (pp. 2857-2862).

The application of near-infrared (NIR) dyes (λ _em > 750 nm) to the analysis of biological samples shows much promise, because the long emission wavelengths of such dyes allow interferences from biomolecule matrices to be minimized. In this paper, a novel NIR dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine (DCDSTCY) has been developed for the spectrophotometric determination of total protein in serum. Under acidic conditions, the binding of DCDSTCY to proteins caused a new peak at 878 nm, the height of which was proportional to the concentration of protein. The linear range of the method was found to be 0.04–0.5 μg mL⁻¹ for bovine serum albumin (BSA) and human serum albumin (HSA), and detection limits of 5 ng mL⁻¹ were obtained for these substances. The maximum binding number of BSA with DCDSTCY was measured to be 133. The method proposed here has been applied to the quantitation of total protein in serum, and recoveries of 96.6–104% were achieved. Figure Near-infrared probe for protein determination

Keywords: Near-infrared (NIR) dye; 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine (DCDSTCY); Spectrophotometry; Bovine serum albumin (BSA); Human serum albumin (HSA)

Sol-gel-based optical sensor for the detection of aqueous amines by Špela Mojca Korent; Aleksandra Lobnik; Gerhard J. Mohr (pp. 2863-2870).

We present an optical sensor for the detection of aqueous amines obtained by incorporating chromoionophore XV (ETH^T 4001) into sol-gel thin films. Acid- and base-catalyzed sol-gel processes were studied to prepare stable ormosil layers using various amounts of organically modified sol-gel precursor such as methyltriethoxysilane (MTriEOS). The sensor layers were coated with a protective layer of microporous white polytetrafluoroethylene (PTFE) in order to prevent interference from ions and ambient light. The measurements were carried out in a flow-through cell in the reflection mode. Acid-catalyzed ormosil layers (pH 1) based on the copolymerization of tetraethoxysilane (TEOS) and MTriEOS did not show any change in signal upon exposure to aqueous amine solutions, while base-catalyzed sensor layers (pH 3 and 13) showed significant changes in signal. The response time (t ₁₀₀) for the base-catalyzed sensor layer L3 (pH 13) upon exposure to different solutions containing 0–608 mmol L⁻¹ aqueous propylamine was 20–30 s, the regeneration time was 70 s and the detection limit was 0.1 mmol L⁻¹. The sensor response was reproducible and reversible. The porous ormosil layers permit dry sensor storage conditions.

Keywords: Optical sensor; Sol-gel; Amine detection

Sol-gel-based optical sensor for the detection of aqueous amines by Špela Mojca Korent; Aleksandra Lobnik; Gerhard J. Mohr (pp. 2863-2870).

Keywords: Optical sensor; Sol-gel; Amine detection

Accurate quantification of freely dissolved organochlorine pesticides in water in the presence of dissolved organic matter using triolein-embedded cellulose acetate membrane by Runhui Ke; Zijian Wang; Shengbiao Huang; Shahamat U. Khan (pp. 2871-2879).

A novel method is described using triolein-embedded cellulose acetate membrane (TECAM) for accurate determination of the freely dissolved fraction of organochlorine pesticides (OCPs) in waters rich in dissolved organic matter (DOM). The performance of the method was tested with an air-bridge system for extracting OCPs from aqueous solutions with and without humic acid. In addition, the partition coefficients between humic acid and water (K _docs) for 20 OCPs were determined by TECAM with negligible depletion extraction. Results show that TECAM predominantly extracts the freely dissolved compounds and its extraction efficiency decreases significantly with an increase in concentration of humic acid in water. The proposed methodology is suitable for facile laboratory K _doc measurement for moderate to high hydrophobic compounds (log K _ow > 4). The linear relationship between log K _ow and log K _doc obtained in this study agrees well with the results reported earlier. The kinetic uptake rate constants (k _us) and TECAM–water partition coefficients (K _TECAMs) for the 20 OCPs were obtained using the controlled laboratory continuous-flow and static exposure system, respectively. These calibration parameters were used in the field experiment to estimate the freely dissolved concentrations of OCPs in the water of Taihu Lake in China. Our results show that TECAM can be used successfully to determine the freely dissolved OCPs in aquatic environments containing DOM, and the method is particularly suited for long-term water sampling. Figure Schematic diagram of water sampling with a triolein-embedded cellulose acetate membrane (TECAM)

Keywords: Organochlorine pesticides; Dissolved organic matter; Humic acid; Passive sampling; Freely dissolved concentration

Methylene blue derivatization then LC–MS analysis for measurement of trace levels of sulfide in aquatic samples by Jeffrey M. Small; Holger Hintelmann (pp. 2881-2886).

The methylene blue method has been widely used for analysis of sulfide for more than 100 years. Direct measurement of methylene blue at nanomolar concentrations is impossible without a preconcentration step, however. In this study the response of LC–MS with electrospray ionization (ESI) to methylene blue was evaluated. HPLC with simple isocratic elution was followed by ESI-MS quantification, which was compared with traditional UV–visible detection. The limit of detection for sulfide was approximately 50 ng L⁻¹, or 1.5 nmol L⁻¹. Analysis time was substantially reduced by use of isocratic elution. Interfering compounds produced by side reactions can be eliminated by use of the mass filter. A polysulfide sample was also analyzed to determine which products are formed and whether or not polysulfides react stoichiometrically with methylene blue reagent. It seems that polysulfides do not react quantitatively with methylene blue and so cannot be quantified reliably by use of this method.

Keywords: Sulfide; Polysulfides; Water; Methylene blue; LC–MS

Methylene blue derivatization then LC–MS analysis for measurement of trace levels of sulfide in aquatic samples by Jeffrey M. Small; Holger Hintelmann (pp. 2881-2886).

Keywords: Sulfide; Polysulfides; Water; Methylene blue; LC–MS

Determination of iprodione in agrochemicals by infrared and Raman spectrometry by Sergio Armenta; Salvador Garrigues; Miguel de la Guardia (pp. 2887-2894).

Two methodologies based on vibrational spectrometry—making use of Fourier transform infrared absorption (FTIR) and Raman spectrometry—were developed for iprodione determination in solid pesticide formulations. The FTIR procedure involved the extraction of iprodione by CHCl₃, and the latter determination involved measuring the peak area between 1450 and 1440 cm⁻¹, corrected using a horizontal baseline defined at 1481 cm⁻¹. FT-Raman determination was performed directly on the powdered solid products, using standard chromatography glass vials as sample cells and measuring the Raman intensity between 1003 and 993 cm⁻¹, with a two-point baseline correction established between 1012 and 981 cm⁻¹. The sensitivities obtained were 0.319 area values g mg⁻¹ for FTIR determination and 5.58 area values g g⁻¹ for FT-Raman. The repeatabilities, taken to be the relative standard deviation of five independent measurements at 1.51 mg g⁻¹ and 10.98% w/w concentration levels, were equal to 0.16% and 0.9% for FTIR and FT-Raman, respectively, and the limits of detection were 0.3 and 0.2% w/w (higher than those obtained for HPLC, 0.016% w/w). FTIR determination provided a sample frequency of 60 h⁻¹, higher than those obtained for the Raman and reference chromatography methods (25 and 8.6 h⁻¹, respectively). On the other hand, the new FT-Raman method eliminates reagent consumption and waste generation, and reduces the need for sample handling and the contact of operator with the pesticide. In spite of their lack of sensitivity, vibrational procedures can therefore provide viable environmentally friendly alternatives to laborious, time- and solvent-consuming reference chromatography methods for quality control in commercially available pesticide formulations.

Keywords: Iprodione; Pesticide formulations; Infrared; Raman; Green analytical method

Determination of iprodione in agrochemicals by infrared and Raman spectrometry by Sergio Armenta; Salvador Garrigues; Miguel de la Guardia (pp. 2887-2894).

Keywords: Iprodione; Pesticide formulations; Infrared; Raman; Green analytical method

Wax components of larval cocoon silk of the hornet Vespa analis Fabricius by Tsunenori Kameda; Toshiharu Akino; Katsura Kojima (pp. 2895-2902).

Wax, 85% of which consists of orthorhombic crystals, has been found in the cocoon of the hornet Vespa analis Fabricius by means of high-resolution ¹³C solid-state nuclear magnetic resonance (NMR). GC–MS analysis revealed the major components of the wax in the cocoon were linear alkenes and alkanes with a total of 23 or 34 carbon atoms. At 40.7 °C a DSC absorption peak and a ¹³C NMR chemical shift change were observed and interpreted as the result of a crystal transition from the orthorhombic to rotator phase of the wax molecules. At 55.5 °C melting of the wax was observed. The amount of crystalline wax deposition varied with the part of the cocoon—crystalline wax was concentrated in the silk sleeve lining the inner wall of each comb cell but there was very little in the silk cap projecting from the end of each cell. Because the wax components of the larval cocoon were almost identical to those of the larval cuticle, despite a slight difference in the profiles, they might have come from the larval cuticle via direct body contact with the cocoon. Figure Cocoon of the hornet Vespa analis Fabricius

Keywords: Solid-state NMR; GC–MS; DSC; Wax; Hornet cocoon silk

Wax components of larval cocoon silk of the hornet Vespa analis Fabricius by Tsunenori Kameda; Toshiharu Akino; Katsura Kojima (pp. 2895-2902).

Keywords: Solid-state NMR; GC–MS; DSC; Wax; Hornet cocoon silk

Precise measurement of ¹H 90° pulse in solid-state NMR spectroscopy for complex and heterogeneous molecular systems by Pellegrino Conte; Alessandro Piccolo (pp. 2903-2909).

The 90° pulse calibration is essential in NMR spectroscopy to prevent artefacts in the liquid state or to enhance cross-polarization efficiency in the solid state. We verified pulse-angle (PA) errors due to circuit impedances in solid-state NMR and suggested a possible solution to prevent the inconvenience of PA errors. The classic pulse sequences used to calibrate ¹H 90° pulse lengths by direct detection of protons or by cross-polarization were modified in order to replace single ¹H pulses with ¹H pulse trains. Pulse trains were found to decrease the effect of PA imperfections in the calibration of basic pulses (i.e. 90° and 180°) for a number of organic substrates. The modified sequences are especially important to rapidly obtain pulse calibration of complex and heterogeneous molecular systems such as humic substances, which usually require a long time when using single ¹H pulses.

Keywords: Cross polarization magic angle spinning NMR; ¹H 90° pulse; Pulse calibration; Circuit impedance; Complex molecular systems

Precise measurement of ¹H 90° pulse in solid-state NMR spectroscopy for complex and heterogeneous molecular systems by Pellegrino Conte; Alessandro Piccolo (pp. 2903-2909).

Keywords: Cross polarization magic angle spinning NMR; ¹H 90° pulse; Pulse calibration; Circuit impedance; Complex molecular systems

Nonequilibrium hollow-fiber liquid-phase microextraction with in situ derivatization for the measurement of triclosan in aqueous samples by gas chromatography–mass spectrometry by Ru-Song Zhao; Jin-Peng Yuan; Hai-Fang Li; Xu Wang; Ting Jiang; Jin-Ming Lin (pp. 2911-2915).

Hollow-fiber liquid-phase microextraction (HF-LPME), a relatively new sample preparation technique, has attracted much interest in the field of environmental analysis. In the current study, a novel method based on hollow-fiber liquid-phase microextraction with in situ derivatization and gas chromatography–mass spectrometry for the measurement of triclosan in aqueous samples is described. Hollow-fiber liquid-phase microextraction conditions such as the type of extraction solvent, the stirring rate, the volume of derivatizing reagent, and the extraction time were investigated. When the conditions had been optimized, the linear range was found to be 0.05–100 μg l⁻¹ for triclosan, and the limit of detection to be 0.02 μg l⁻¹. Tap water and surface water samples collected from our laboratory and Wohushan reservoir, respectively, were successfully analyzed using the proposed method. The recoveries from the spiked water samples were 83.6 and 114.1%, respectively; and the relative standard deviation (RSD) at the 1.0 μg l⁻¹ level was 6.9%.

Keywords: Triclosan; Hollow-fiber liquid-phase microextraction; Gas chromatography–mass spectrometry; In situ derivatization

Online phototransformation–flow injection chemiluminescence determination of triclosan by Shujuan Song; Qi Jun Song; Zhongliang Chen (pp. 2917-2922).

A highly selective and sensitive chemiluminescence method for the determination of triclosan is proposed. The method is based on the phototransformation of triclosan to a light-emitting precursor in the presence of fluorescein in alkaline medium and the chemiluminescence reaction is then triggered by strong base or oxidants such as N-bromosuccinimide. Based on this reaction an online phototransformation–flow injection manifold was developed, in which the photoreactor comprises a 150-cm-long × 0.8-mm-i.d. piece of PTFE tubing coiled around a 25-W fluorescent lamp, and the phototransformed products were then injected into a carrier stream of borate buffer. After mixing with the oxidant stream the produced light was detected by a photomultiplier. A wide calibration range from 8.0 × 10⁻⁸ to 1.0 × 10⁻⁴ mol L⁻¹ was obtained under the optimized conditions, and the detection limit was as low as 5.0 × 10⁻⁸ mol L⁻¹. The whole process of analysis, including the online phototransformation and subsequent chemiluminescence detection, could be completed in 6 min. Most of the foreign substances tested showed high tolerance levels, and the proposed method was directly applied to the determination of triclosan in toothpaste samples without any pre-separation procedure. Figure Schematic representation of the phototransformation of triclosan and subsequent chemiluminescence reaction

Keywords: Triclosan; Singlet oxygen; Phototransformation; Chemiluminescence

Online phototransformation–flow injection chemiluminescence determination of triclosan by Shujuan Song; Qi Jun Song; Zhongliang Chen (pp. 2917-2922).

Keywords: Triclosan; Singlet oxygen; Phototransformation; Chemiluminescence

Microwave-assisted extraction (MAE) for the determination of polybrominated diphenylethers (PBDEs) in sewage sludge by Mari Shin; M. Lewina Svoboda; Patricia Falletta (pp. 2923-2929).

An efficient microwave-assisted extraction (MAE) method has been developed and evaluated for the quantification of eight major polybrominated diphenylethers (PBDEs) in sewage sludge. The PBDEs were extracted from wet and dry sludge in a microwave extraction unit using a hexane/acetone mixture for 35 min at a controlled temperature of 130 °C. The extract was concentrated, cleaned up on a silica gel column, and analyzed by gas chromatography/mass spectrometry (GC/MS) in the negative chemical ionization (NCI) mode. The MAE procedure exhibited higher extraction efficiency, specifically for BDE (brominated diphenylether) 209, than the conventional Soxhlet extraction. The test congeners were clearly separated under specific instrumental operating conditions, at a source temperature of 230 °C and a column length of 20 m. The present analytical method showed recovery efficiencies ranging from 80 to 110% when applied to the PBDE-free sludge spiked with eight PBDE congeners. The efficiency of the MAE method was confirmed using sludge obtained from four sewage treatment plants (STPs). The results indicate that BDE 47, 99, and 209 are the most abundant congeners present in these sewage sludges, which is consistent with previous reports.

Keywords: Polybrominated diphenylethers; Microwave-assisted extraction; Sewage sludge; Soxhlet extraction

Microwave-assisted extraction (MAE) for the determination of polybrominated diphenylethers (PBDEs) in sewage sludge by Mari Shin; M. Lewina Svoboda; Patricia Falletta (pp. 2923-2929).

Keywords: Polybrominated diphenylethers; Microwave-assisted extraction; Sewage sludge; Soxhlet extraction

Assessment of metastable atom bombardment (MAB) ionization mass spectrometry for the fast determination of heterocyclic aromatic amines in cooked meat by E. Jamin; S. Chevolleau; C. Touzet; J. Tulliez; L. Debrauwer (pp. 2931-2941).

An investigation of metastable atom bombardment (MAB) ionization mass spectrometry for the fast characterization of mutagenic/carcinogenic heterocyclic aromatic amines (HAAs) formed during heating processes of meats is presented. The aim of our study was to use the selective ionization of MAB to develop a detection method for HAAs in non-purified meat extracts, thus avoiding purification and concentration steps and reducing analysis time. Sample introduction into the MAB ion source was achieved by pyrolysis, allowing the direct and fast insertion of complex food extracts into the mass spectrometer. Analysis conditions were optimized on standard HAAs by using different ionization gases for the MAB process. Metastable nitrogen was selected as the best MAB gas for the analysis of HAAs. Ionization selectivity is shown by the detection of heterocyclic amines in non-purified chicken meat extracts spiked with HAAs. A quantitative approach is also presented by using pyrograms as chromatograms for quantification purposes. HAAs determination using Py-MAB-ToF was finally performed on cooked chicken breast extracts and compared to an LC-APCI-MS/MS method. Although Py-MAB-ToF sensitivity remains to be improved in the present state of development of our prototype device, only 2 h from the cooking were required to obtain quantitative results in good agreement with HAAs concentrations measured by LC-MS/MS in 36 h. Figure Experimental set-up for pyrolysis-MAB-ToF mass spectrometry experiments.

Keywords: Heterocyclic aromatic amines; Chicken; Metastable atom bombardment; Pyrolysis; Mass spectrometry

Application of a sensitive fluorometric HPLC assay to determine the sialic acid content of infant formulas by María J. Martín; Enrique Vázquez; Ricardo Rueda (pp. 2943-2949).

The developing human brain requires high amounts of sialic acids. While human milk is very rich in sialic acids, cow’s milk based infant formulas provide lower amounts of sialic acids, and sialic acids are absent in soy milk based formulas. This has prompted interest in the supplementation of formulas with sialic acids, either free or bound to glycoconjugates. In order for fortification of infant formulas with sialic acids to be appropriate for the developmental needs of the infant, an accurate quantitation of sialic acid content of infant formulas through a reliable and easy-to-use method is, therefore, of great interest to industry. In the present method, we describe the application of one of the most widely used analytical techniques to the quantitation of sialic acids in infant formulas. Briefly, sialic acids are hydrolyzed from glycoconjugates, derivatized using 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride (DMB), and separated using reversed-phase high-performance liquid chromatography. The method fulfilled the established criteria for validation, with an interassay standard deviation of less than 5%, accuracy greater than 97%, and surrogate recovery between 98 and 104%. An investigation of the ruggedness of the method identified two key criteria: both standards and samples must be subjected to the same temperature and pH conditions for an accurate quantitation; and prolonged storage (more than 2 days) of the DMB reagent and derivatives must be avoided. In conclusion, this method is specific, straightforward, and accurate and can be easily performed in a quality-assurance laboratory to track the level of sialic acid in formulas that contain both inherent and fortified amounts of sialic acids. Figure Infant formula and HPLC vials used for the sialic acid quantitation

Keywords: Infant formula; Sialic acids; High-performance liquid chromatography; 1,2-Diamino-4,5-methylenedioxybenzene dihydrochloride

Application of a sensitive fluorometric HPLC assay to determine the sialic acid content of infant formulas by María J. Martín; Enrique Vázquez; Ricardo Rueda (pp. 2943-2949).

Keywords: Infant formula; Sialic acids; High-performance liquid chromatography; 1,2-Diamino-4,5-methylenedioxybenzene dihydrochloride

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Analytical and Bioanalytical Chemistry (v.387, #8)

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Analytical and Bioanalytical Chemistry (v.387, #8)