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Analytical and Bioanalytical Chemistry (v.392, #5)

Spectroscopy challenge 13 by Reinhard Meusinger (pp. 767-770).

Solution to nascent hydrogen challenge by Juris Meija; Alessandro D’Ulivo (pp. 771-772).

32nd International Symposium on Capillary Chromatography and 5th GC×GC Symposium by Hannelore Kaspar (pp. 773-774).

Johanna Sabine Becker: Inorganic mass spectrometry. Principles and applications by Norbert Jakubowski (pp. 775-776).

Steffen Hardt and Friedhelm Schönfeld (Eds.): Microfluidic technologies for miniaturized analysis systems by Alexandra Ros (pp. 777-778).

Damià Barceló (Ed.): Fuel oxygenates by L. Reijnders (pp. 779-780).

DGMS 2008: The 41st Annual Meeting of the German Society for Mass Spectrometry by Bernhard Spengler (pp. 781-782).

is full Professor of Analytical Chemistry at the Justus Liebig University of Giessen, Germany. He has been involved in developments of mass spectrometry techniques and methods for more than 20 years. After his PhD in 1988 in the group of Franz Hillenkamp he joined R.J. Cotter at the Johns Hopkins University, Baltimore, as a postdoc. He received his Habilitation in Biophysics at the Institute of Laser Medicine, University of Duesseldorf. In 1998 he became Professor of Physical Chemistry at the Bayerische Julius-Maximilians-Universität Würzburg. In 2000 he joined the Justus Liebig University of Giessen. As a member of the large instrument panel of the DFG he is responsible for the field of MS, LC, and related equipment. He is recipient of the Bennigsen-Foerder Award of the state of North Rhine-Westphalia. In 2006 he received the Life Science Award of the Deutsche Gesellschaft für Massenspektrometrie..

Mass spectrometry for monitoring protease reactions by H. Schlüter; D. Hildebrand; C. Gallin; A. Schulz; J. Thiemann; M. Trusch (pp. 783-792).

More than 560 genes are annotated as proteases in the human genome. About half of the genes are not or are only marginally characterized. Over the past decade, mass spectrometry has become the basis for proteomics, especially for protein identification, performed in a high-throughput manner. This development was also very fruitful for exploring the complex systems associated with protease functions, as briefly reviewed here. Mass spectrometry is an ideal tool for monitoring protease reactions, as will be highlighted in this review.

Keywords: Bioanalytical methods; Enzymes; Mass spectrometry

Grazing incidence surface-induced dissociation: molecules sliding along a surface by Alexander Zulauf; Lothar Schmidt; Hartmut Jungclas (pp. 793-796).

Principles of grazing incidence SID and a brief overview of previous works are summarized and the experimental setup for grazing incidence SID experiments is described. New results with fullerene C₆₀ are presented; these demonstrate that grazing incidence SID is not a special case of the conventional SID.

Keywords: Grazing incidence SID; Time-of-flight; MALDI; Fullerenes; Peptides

LC-MS-MS aboard ship: tandem mass spectrometry in the search for phycotoxins and novel toxigenic plankton from the North Sea by Bernd Krock; Urban Tillmann; Uwe John; Allan Cembella (pp. 797-803).

Phycotoxins produced by various species of toxigenic microalgae occurring in the plankton are a global threat to the security of seafood resources and the health of humans and coastal marine ecosystems. This has necessitated the development and application of advanced methods in liquid chromatography coupled to mass spectrometry (LC-MS) for monitoring of these compounds, particularly in plankton and shellfish. Most such chemical analyses are conducted in land-based laboratories on stored samples, and thus much information on the near real-time biogeographical distribution and dynamics of phycotoxins in the plankton is unavailable. To resolve this problem, we conducted ship-board analysis of a broad spectrum of phycotoxins collected directly from the water column on an oceanographic cruise along the North Sea coast of Scotland, Norway, and Denmark. We equipped the ship with a triple-quadrupole linear ion-trap hybrid LC-MS-MS system for detection and quantitative analysis of toxins, such as domoic acid, gymnodimine, spirolides, dinophysistoxins, okadaic acid, pectenotoxins, yessotoxins, and azaspiracids (AZAs). We focused particular attention on the detection of AZAs, a group of potent nitrogenous polyether toxins, because the culprit species associated with the occurrence of these toxins in shellfish has been controversial. Marine toxins were analyzed directly from size-fractionated plankton net tows (20 μm mesh size) and Niskin bottle samples from discrete depths, after rapid methanolic extraction but without any further clean-up. Almost all expected phycotoxins were detected in North Sea plankton samples, with domoic acid and 20-methylspirolide G being most abundant. Although AZA was the least abundant of these toxins, the high sensitivity of the LC-MS-MS enabled detailed quantification, indicating that the highest amounts of AZA-1 were present in the southern Skagerrak in the 3–20 μm size-fraction. The direct on-board toxin measurements enabled isolation of plankton from stations with high AZA-1 levels and from the most suspicious size-fraction, i.e. most likely to contain the AZA-producer. A large number (>100) of crude cultures were established by serial dilution and later screened for the presence of AZAs after several weeks growth. From one crude culture containing AZA, a small dinoflagellate was subsequently isolated and brought into pure culture. We have thus proved that even sophisticated mass spectrometers can be operated in ship laboratories without any limitation caused by vibrations of the ship’s engine or by wave movement during heavy seas at wind forces up to nine Beaufort. On-board LC–MS–MS is a valuable method for near real-time analysis of phycotoxins in plankton for studies on bloom dynamics and the fate of toxins in the food web, and for characterization and isolation of putatively toxigenic organisms.

Keywords: North Sea; Plankton; Azaspiracids; AZA; Phycotoxins; Shellfish toxin; LC-MS-MS

Separation and identification of trinucleotide–melphalan adducts from enzymatically digested DNA using HPLC–ESI–MS by Dalia Mohamed; Michael Linscheid (pp. 805-817).

Melphalan is a bifunctional alkylating agent that covalently binds to the nucleophilic sites present in DNA. In this study we investigated oligonucleotides prepared enzymatically from DNA modified with melphalan. Calf thymus DNA was incubated in-vitro with melphalan and the resulting modifications were enzymatically cleaved by means of benzonase and nuclease S1. Efficient sample preconcentration was achieved by solid-phase extraction, in which phenyl phase cartridges resulted in better recovery of the modified species than C₁₈. The applied enzymatic digestion time resulted in production of trinucleotide adducts which were efficiently separated and detected by use of reversed-phase HPLC coupled to an ion-trap mass spectrometer with electrospray ionization. It was assumed that melphalan could act as both a monofunctional and bifunctional alkylating agent. Mono-alkylated adducts were much more abundant, however, and the alkylation site was located on the nucleobases. On the other hand, we unequivocally identified cross-link formation in DNA, even though at low abundance and only a few adduct types were detected. Figure Different Alkylation reactions of Melphalan with DNA

Keywords: Melphalan; Nitrogen mustards; DNA adducts; LC–MS–MS; Fragmentation

Separation and characterization of oxaliplatin dinucleotides from DNA using HPLC-ESI ion trap mass spectrometry by Shereen Mowaka; Michael Linscheid (pp. 819-830).

Oxaliplatin is a third-generation platinum complex, and has a broad spectrum of antitumor activity. Such platinum complexes with the DACH carrier ligand have recently received increasing attention since they show efficacy against cisplatin-resistant cell lines. As the foremost indication of antitumor activity of platinum drugs is the formation of adducts with genomic DNA, calf thymus DNA-oxaliplatin adducts were the major target in this study. Calf thymus DNA was incubated with oxaliplatin, resulting in the formation of a large number of platinum-DNA adducts. Treated DNA was digested into the dinucleotides with a combination of enzymes, namely, benzonase, alkaline phosphatase, and nuclease S1. Using a high-performance liquid chromatography, we carried out the separation of individual platinum-DNA adducts which were concurrently identified using electrospray ionization ion trap mass spectrometry (MS). Both 1,2-intrastrand and 1,2-interstrand cross-linked adducts were found; however, those of the intrastrand nature have a considerably higher abundance than those of the interstrand cross-links. Among them, d(GpG)-oxaliplatin was the most abundant bifuctional adduct. To a lesser extent, a few monofunctional adducts were detected as well. MS n experiments served to ascertain the detailed structures of oxaliplatin adducts of dinucleoside monophosphates and of dinucleotides. Figure Three-dimensional view of the d(GpG)-Pt(DACH) adduct of m/z 902 (a) and of the d(ApG)-Pt(DACH) adduct of m/z 886 (b). DACH 1,2-diaminocyclohexane, green phosphorus, red oxygen, light blue carbon, dark blue nitrogen, gray platinum

Keywords: Oxaliplatin; DNA adducts; Benzonase; Nuclease S1; HPLC-ESI-MS; Fragmentation

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation by Michael Mormann; Johannes Eble; Christian Schwöppe; Rolf M. Mesters; Wolfgang E. Berdel; Jasna Peter-Katalinić; Gottfried Pohlentz (pp. 831-838).

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.

Keywords: Inter-and intramolecular disulfide bridge; Underivatised peptides; Low-energy collision-induced dissociation; Asymmetric disulfide-bridge cleavage

Fourier transform ion cyclotron resonance mass spectrometry in the speciation of high molecular weight sulfur heterocycles in vacuum gas oils of different boiling ranges by Saroj K. Panda; Wolfgang Schrader; Jan T. Andersson (pp. 839-848).

The analysis of sulfur aromatics in vacuum gas oils (VGO) distilled from an Iranian light crude oil is discussed. The VGOs were fractionated into three boiling ranges, 390–460, 460–520, and 520–550 °C, and were analyzed using liquid chromatographic separation on a Pd(II)-bonded stationary phase followed by identification with electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). It was possible to detect a large number of thiophenes, including a substantial number of isomers, in the three VGO fractions. Separation on the palladium phase and inclusion of sulfur-selective derivatization makes electrospray ionization of these nonpolar compounds possible. An elemental composition can be assigned to a large number of S₁ compounds without ambiguity in the presence of abundant hydrocarbons. With an increase in boiling temperature, an increase in the size of the aromatic system and the number of side chain carbon atoms was observed. In addition, the masses of higher magnitude shifted toward larger aromatic systems with an increase in boiling range. A comparison of FT-ICR MS and comprehensive gas chromatography is also given.

Keywords: Fourier transform ion cyclotron resonance mass spectrometry; Crude oil analysis; Liquid chromatography; Electrospray; Sulfur

Analysis of stem cell lipids by offline HPTLC-MALDI-TOF MS by Beate Fuchs; Jürgen Schiller; Rosmarie Süß; Matthias Zscharnack; Augustinus Bader; Peter Müller; Martin Schürenberg; Michael Becker; Detlev Suckau (pp. 849-860).

MALDI-TOF MS is traditionally used for “proteomics”, but is also a useful tool for lipid analysis. Depending on the applied matrix, however, some lipid classes are more sensitively detected than other ones and this may even lead to suppression effects if complex mixtures are analyzed. Therefore, a previous separation into the individual lipid classes is necessary. Using artificial lipid mixtures or easily available tissue extracts, it has been already shown that HPTLC-(High Performance Thin-Layer Chromatography)-separated lipids can be conveniently analyzed by MALDI-TOF MS directly on the TLC plate. Here we present an initial TLC-MALDI study of the lipid composition of ovine mesenchymal stem cells. Due to the complex composition of these cells, data are also compared to lipids extracted from human erythrocytes. It will be shown that even very minor lipid classes can be easily detected and with much higher sensitivity than by common staining protocols. Additionally, MS images of the developed TLC plates will be shown and potential applications, new methods of data analysis as well as problems discussed. Figure

Keywords: MALDI-TOF MS; High Performance Thin-Layer Chromatography (HPTLC); Mesenchymal stem cells; Lipids; MS imaging; Erythrocytes

Conducting polymer-based impedimetric aptamer biosensor for in situ detection by Wei Liao; Brad A. Randall; Nicolas A. Alba; Xinyan Tracy Cui (pp. 861-864).

We have successfully developed the first prototype of an electrochemical protein biosensor using polypyrrole as the substrate and doped aptamer as the probe. The sensitivity for a specific target is 10 ng/ml. Two targets, platelet-derived growth factor and immunoglobulin E, have been tested. In both cases the sensor exhibited high specificity and the signal was found to increase as the target concentration increased.

Keywords: Aptamer biosensor; Platelet-derived growth factor; Immunoglobulin E; In situ monitoring. Electrochemical impedance spectroscopy

New off-line aircraft instrumentation for non-methane hydrocarbon measurements by Joelle Bechara; Agnès Borbon; Corinne Jambert; Pascal E. Perros (pp. 865-876).

New off-line instrumentation was developed to implement measurements of non-methane hydrocarbons (NMHC) on (French) research aircraft. NMHC are collected on multisorbent tubes by AMOVOC (Airborne Measurements Of Volatile Organic Compounds), a new automatic sampler. AMOVOC is a versatile and portable sampler targeting a wide range of NMHC at high frequency (sampling time of 10 min). Multisorbent tubes are analyzed on the ground by short-path thermal desorption coupled with gas chromatography and mass spectrometry. The development and optimization of both NMHC sampling and analysis are reported here. On the one hand, the paper points out technical choices that were made according to aircraft constraints and avoiding sample loss or contamination. On the other hand, it describes analytical optimization, tube storage stability, and moisture removal. The method shows high selectivity, sensitivity (limit of detection less than 10 ppt) and precision (less than 24%). Finally, NMHC data collected on French aircraft during the African Monsoon Multidisciplinary Analysis campaign are reported for the first time. The results highlight instrumentation validity and protocol efficiency for NMHC measurements in the lower and upper troposphere.

Keywords: Airborne measurements; Non-methane hydrocarbons; Multisorbent tubes; Thermal desorption; Gas chromatography–mass spectrometry; African Monsoon Multidisciplinary Analysis

Magnetic microsphere-based methods to study the interaction of teicoplanin with peptides and bacteria by Menake E. Piyasena; Lilian J. Real; Rochelle A. Diamond; H. Howard Xu; Frank A. Gomez (pp. 877-886).

Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many Gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal d-Ala-d-Ala intermediates in the peptidoglycan of the cell wall of Gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two d-Ala-d-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled d-Ala-d-Ala-d-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 × 10⁻⁶ and 3.0 × 10⁻⁶ mol L⁻¹, respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of Gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide–antibiotic interaction.

Keywords: Bioanalytical methods; Magnetic microspheres; Teicoplanin; Fluorescence; Microfluidics

Addition of gold nanoparticles to real-time PCR: effect on PCR profile and SYBR Green I fluorescence by Adam L. Haber; Kate R. Griffiths; Åsa K. Jamting; Kerry R. Emslie (pp. 887-896).

Real-time quantitative polymerase chain reaction (qPCR) is the industry standard technique for the quantitative analysis of nucleic acids due to its unmatched sensitivity and specificity. Optimisation and improvements of this fundamental technique over the past decade have largely consisted of attempts to allow faster and more accurate ramping between critical temperatures by improving assay reagents and the thermal geometry of the PCR chamber. Small gold nanoparticles (Au-NPs) have been reported to improve PCR yield under fast cycling conditions. In this study, we investigated the effect of Au-NPs on optimised real-time qPCR assays by amplifying DNA sequences from genetically modified canola in the presence and absence of 0.9 nM Au-NPs of diameter 12 ± 2nm. Contrary to expectations, we found that Au-NPs altered the PCR amplification profile when using a SYBR Green I detection system due to fluorescence quenching; furthermore, high-resolution melt (HRM) analysis demonstrated that Au-NPs destabilised the double-stranded PCR product. The results indicate that effects on the assay detection system must be carefully evaluated before Au-NPs are included in any qPCR assay. Figure Raw amplification profiles in the presence and absence of gold nanoparticles

Keywords: Efficiency; Fluorescence quenching; Gold nanoparticles; PCR; Polymerase chain reaction; Real-time PCR

Acoustic determination of performance and equivalence of plasminogen activators by Mirnader Ghazali; Gordon L. Hayward (pp. 897-902).

A reliable method for the measurement of different plasminogen activators is of great interest for both manufacturing and clinical medicine. A one-step assay based on a thickness shear mode acoustic sensor has been developed for this purpose. Two separate mixtures of substrates (fibrinogen and plasminogen) and enzymes (thrombin and the plasminogen activator) were mixed, and placed on the acoustic sensor surface. During the assay, the resonant frequency of a quartz crystal oscillating in the thickness shear mode was measured and used to find a characteristic clot dissolution time, from the sample addition to the time at the maximum dissolution rate. Calibrations of the acoustic assay were done for tissue-type plasminogen activator (t-PA) as well as for the other plasminogen activators: urokinase (u-PA); streptokinase (SK) and staphylokinase (SAK). All gave relative standard deviations of about 12%. Since the same method was used for all of the activators, their activities were compared, resolving the differences between their unit definitions. Linear relationships were found between urokinase and streptokinase which activate plasminogen directly and between t-PA and staphylokinase which require fibrin as a cofactor. The relationship between the groups was found to curve, indicating the difference between the two mechanisms. The acoustic method, therefore, may be used as a rapid and cost-effective reference method for the standardization and comparison of different plasminogen activators.

Keywords: Acoustic assay; Unit equivalence; Plasminogen activators; Thickness shear mode sensor

Development and validation of a liquid chromatography–tandem mass spectrometry assay for the simultaneous quantification of buprenorphine, norbuprenorphine, and metabolites in human urine by Sherri L. Kacinko; Marta Concheiro-Guisan; Diaa M. Shakleya; Marilyn A. Huestis (pp. 903-911).

A liquid chromatography–tandem mass spectrometry method for the simultaneous quantification of buprenorphine (BUP), norbuprenorphine (NBUP), buprenorphine glucuronide (BUP-Gluc), and norbuprenorphine glucuronide (NBUP-Gluc) in human urine was developed and fully validated. Extensive endogenous and exogenous interferences were evaluated and limits of quantification were identified empirically. Analytical ranges were 5–1,000 ng/mL for BUP and BUP-Gluc and 25–1,000 ng/mL for NBUP and NBUP-Gluc. Intra-assay and interassay imprecision were less than 17% and recovery was 93–116%. Analytes were stable at room temperature, at 4 °C, and for three freeze–thaw cycles. This accurate and precise assay has sufficient sensitivity and specificity for urine analysis of specimens collected from individuals treated with BUP for opioid dependence.

Keywords: Buprenorphine; Norbuprenorphine; Buprenorphine glucuronide; Norbuprenorphine glucuronide; Urine; Liquid chromatography–tandem mass spectrometry

Electrosynthesized, non-conducting films of poly(2-naphthol): electrochemical and XPS investigations by Rosanna Ciriello; Antonio Guerrieri; Filomena Pavese; Anna Maria Salvi (pp. 913-926).

Advanced biosensors are frequently based on electrosynthesized polymeric films. In this context, the electrosynthesis mechanism underlying the electrochemical oxidation of 2-naphthol (2-NAP) in phosphate buffer at pH 7 on Pt electrodes has been investigated. The voltammetric behaviour suggested the formation of a non-conducting polymer (poly(2-NAP)) through an irreversible electrochemical process complicated by 2-NAP adsorption and fast electrode passivation. Repeat experiments showed the passive films to be strongly adherent to the Pt surface with thicknesses of approximately 10 nm, as estimated by in-situ electrochemical quartz crystal microbalance (EQCM) measurements and by X-ray photoelectron spectroscopy (XPS). The polymer structure was then investigated by XPS, which gave evidence of the presence of naphthalene rings bonded through poly(oxide) groups (C–O–C) and of quinonoid groups, probably present as the ends of polymeric chains. The polymer repeat unit and terminal groups derived by XPS analysis are in accordance with electrochemical results and with synthesis routes reported for phenol-derived compounds in aqueous solution. XPS also gave evidence of a large excess of oxygen, probably arising from water molecules entrapped by the polymeric chains, as suggested by angle-resolved XPS and thermal treatment of poly(2-NAP)/Pt film under ultra-high vacuum (UHV).

Keywords: 2-Naphthol; Electrochemical polymerization; Passivating film; Biosensors; XPS analysis

A new solid-phase extraction disk based on a sheet of single-walled carbon nanotubes by Hong Yun Niu; Ya Qi Cai; Ya Li Shi; Fu Sheng Wei; Jie Min Liu; Gui Bin Jiang (pp. 927-935).

A new kind of solid-phase extraction disk based on a sheet of single-walled carbon nanotubes (SWCNTs) is developed in this study. The properties of such disks are tested, and different disks showed satisfactory reproducibility. One liter of aqueous solution can pass through the disk within 10–100 min while still allowing good recoveries. Two disks (DD-disk) can be stacked to enrich phthalate esters, bisphenol A (BPA), 4-n-nonylphenol (4-NP), 4-tert-octylphenol (4-OP) and chlorophenols from various volumes of solution. The results show that SWCNT disks have high extraction ability for all analytes. The SWCNT disk can extract polar chlorophenols more efficiently than a C₁₈ disk from water solution. Unlike the activated carbon disk, analytes adsorbed by the new disks can be eluted completely with 8–15 mL of methanol or acetonitrile. Finally, the DD-disk system is used to pretreat 1000-mL real-world water samples spiked with BPA, 4-OP and 4-NP. Detection limits of 7, 25, and 38 ng L⁻¹ for BPA, 4-OP, and 4-NP, respectively, were achieved under optimized conditions. The advantages of this new disk include its strong adsorption ability, its high flow rate and its easy preparation.

Keywords: Solid-phase extraction disk; Single-walled carbon nanotube sheet; C₁₈ disk; Activated carbon disk; Large-volume water sample

Selective isolation of acidic proteins with a thin layer of multiwalled carbon nanotubes functionalized with polydiallyldimethylammonium chloride by Zhuo Du; Yongliang Yu; Jianhua Wang (pp. 937-946).

The selective isolation of acidic proteins using a thin layer of multiwalled carbon nanotubes (MWNTs) functionalized with polydiallyldimethylammonium chloride (PDDA) was demonstrated. A certain amount (20 ml) of a suspension of PDDA-functionalized MWNTs that had been well dispersed by sonication was filtered through an MF-Millipore membrane with a pore aperture of 1.2 μm, and a uniform layer of PDDA-MWNT composites with a thickness of ca. 5 μm formed on the membrane. A 4 × 1 cm piece of the obtained membrane was supported by a stainless steel wire mesh and was then sandwiched between two PTFE films with grooved flow-through channels to form an extraction module. This module with a flow inlet and outlet was incorporated into a sequential injection system for performing the on-line separation and preconcentration of acidic protein, i.e., bovine serum albumin (BSA), and the BSA retained on the layer was eluted with a citrate buffer used as stripping reagent. In addition to a significant reduction in flow resistance, a dynamic sorption capacity of 3.8 mg mg⁻¹ or 1.4 mg cm⁻² for BSA was achieved using the layer-based system—a 146-fold improvement over that obtained using a packed microcolumn mode. A sample volume of 2.0 ml yielded an enrichment factor of 17, a retention efficiency of 100% and a recovery of 95%, along with a sampling frequency of 20 h⁻¹ and a RSD value of 2.8% at 25 μg ml⁻¹ for BSA. The practical applicability of the system was demonstrated by isolating acidic proteins (especially human serum albumin) from whole blood. Figure Selective isolation of acidic proteins with a composite thin layer of multi-walled carbon nanotubes functionalized with poly-diallyldimethylammonium chloride

Keywords: Multiwalled carbon nanotubes; Membrane; Functionalization; Polyelectrolyte; Acidic protein; Extraction

Analysis of meloxicam by high-performance liquid chromatography with cloud-point extraction by Haixia Zhang; Hoo-Kyun Choi (pp. 947-953).

A simple cloud-point extraction method for the determination of meloxicam in human serum was developed. Meloxicam was extracted from serum sample after adding 1 mL of 3% (v/v) Triton X-114 aqueous solution in the presence of 1M HCl and 60 mg NaCl. The meloxicam, present in the surfactant-rich phase, was enriched again with acetonitrile. Tenoxicam was used as the external standard. The separation was achieved on a C18 analytical column with a mobile phase consisting of aqueous acetic acid (1%, v/v) and acetonitrile (54:46, v/v). UV detection was performed at 360 nm. The response was linear over the range 45–2000 ng mL⁻¹ in human serum, and intra- and interday precisions of less than 15.0% were obtained. The relative error was within ±3.0%. The recoveries of meloxicam were larger than 92.0%. The method was compared with liquid–liquid extraction. The results showed that the new method has a considerable LOQ and higher recoveries but poorer precision than liquid–liquid extraction, which exhibited poor recoveries of less than 86.0%, precisions of less than 5.0% and relative errors of less than 7.0%. The method was used for the determination of meloxicam in healthy human volunteers.

Keywords: Meloxicam; Tenoxicam; Cloud-point extraction; High-performance liquid chromatography

Evaluation of a multiresidue method for measuring fourteen chemical groups of pesticides in water by use of LC-MS-MS by J. J. Carvalho; P. C. A. Jerónimo; C. Gonçalves; M. F. Alpendurada (pp. 955-968).

European Council Directive 98/83/EC on the quality of water intended for human consumption brought a new challenge for water-quality control routine laboratories, mainly on pesticides analysis. Under the guidelines of ISO/IEC 17025:2005, a multiresidue method was developed, validated, implemented in routine, and studied with real samples during a one-year period. The proposed method enables routine laboratories to handle a large number of samples, since 28 pesticides of 14 different chemical groups can be quantitated in a single procedure. The method comprises a solid-phase extraction step and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS-MS). The accuracy was established on the basis of participation in interlaboratory proficiency tests, with encouraging results (majority |z-score| <2), and the precision was consistently analysed over one year. The limits of quantitation (below 0.050 μg L⁻¹) are in agreement with the enforced threshold value for pesticides of 0.10 μg L⁻¹. Overall method performance is suitable for routine use according to accreditation rules, taking into account the data collected over one year. Figure Simultaneous SPE extraction system for high thoughput analysis

Keywords: Pesticides; Water; Multiresidue; Solid-phase extraction; Liquid chromatography-mass spectrometry; Uncertainties

Simple and rapid method for the determination of ethylenebisdithiocarbamate fungicides in fruits and vegetables using liquid chromatography with tandem mass spectrometry by Tadashi Hayama; Makoto Takada (pp. 969-976).

A simple and rapid method for determining ethylenebisdithiocarbamates (EBDCs; mancozeb, maneb, and zineb) in fruits and vegetables is described. EBDCs are transformed into dimethylethylenebisdithiocarbamate (EBDC-dimethyl) by methylation after their decomposition with ethylenediaminetetraacetic acid (EDTA). These processes were performed simultaneously in this method. Dimethyl sulfate was used as the methylation reagent, and acetonitrile extracts obtained from partitioning with anhydrous magnesium sulfate and sodium chloride were subjected to dispersive solid-phase extraction with the primary secondary amine sorbent. Liquid chromatography with tandem mass spectrometry in the positive heated-electrospray ionization mode was used for the determination of EBDC-dimethyl produced from EBDCs. The method was validated at levels of 10, 50, and 100 ng g⁻¹ maneb as a representative EBDC. The recoveries of the present method were between 71 and 101%. The limits of detection and quantification were 0.24 and 0.8 ng g⁻¹ maneb, respectively.

Keywords: Ethylenebisdithiocarbamates; Fruits and vegetables; Dimethyl sulfate; Partitioning; Liquid chromatography with tandem mass spectrometry

Optimisation of the extraction of olive (Olea europaea) leaf phenolics using water/ethanol-based solvent systems and response surface methodology by Stefania Mylonaki; Elias Kiassos; Dimitris P. Makris; Panagiotis Kefalas (pp. 977-985).

An experimental setup based on a 2³ full-factorial, central-composite design was implemented with the aim of optimising the recovery of polyphenols from olive leaves by employing reusable and nontoxic solutions composed of water/ethanol/citric acid as extracting media. The factors considered were (i) the pH of the medium, (ii) the extraction time and (iii) the ethanol concentration. The model obtained produced a satisfactory fit to the data with regard to total polyphenol extraction (R ² = 0.91, p = 0.0139), but not for the antiradical activity of the extracts (R ² = 0.67, p = 0.3734). The second-order polynomial equation obtained after analysing the experimental data indicated that ethanol concentration and time mostly affected the extraction yield, but that increased pH values were unfavourable in this regard. The maximum theoretical yield was calculated to be 250.2 ± 76.8 mg gallic acid equivalent per g of dry, chlorophyll-free tissue under optimal conditions (60% EtOH, pH 2 and 5 h). Liquid chromatography–electrospray ionisation mass spectrometry of the optimally obtained extract revealed that the principal phytochemicals recovered were luteolin 7-O-glucoside, apigenin 7-O-rutinoside and oleuropein, accompanied by smaller amounts of luteolin 3′,7-O-diglucoside, quercetin 3-O-rutinoside (rutin), luteolin 7-O-rutinoside and luteolin 3′-O-glucoside. Simple linear regression analysis between the total polyphenol and antiradical activity values gave a low and statistically insignificant correlation (R ² = 0.273, p > 0.05), suggesting that it is not the sheer amount of polyphenols that provides high antioxidant potency; instead, this potency is probably achieved through interactions among the various phenolic constituents.

Keywords: Antiradical activity; Olive leaf; Olea europaea ; Polyphenols; Response surface

Colorimetric method for enzymatic screening assay of ATP using Fe(III)-xylenol orange complex formation by Akihiko Ishida; Yasuko Yamada; Tamio Kamidate (pp. 987-994).

In hygiene management, recently there has been a significant need for screening methods for microbial contamination by visual observation or with commonly used colorimetric apparatus. The amount of adenosine triphosphate (ATP) can serve as the index of a microorganism. This paper describes the development of a colorimetric method for the assay of ATP, using enzymatic cycling and Fe(III)-xylenol orange (XO) complex formation. The color characteristics of the Fe(III)-XO complexes, which show a distinct color change from yellow to purple, assist the visual observation in screening work. In this method, a trace amount of ATP was converted to pyruvate, which was further amplified exponentially with coupled enzymatic reactions. Eventually, pyruvate was converted to the Fe(III)-XO complexes through pyruvate oxidase reaction and Fe(II) oxidation. As the assay result, yellow or purple color was observed: A yellow color indicates that the ATP concentration is lower than the criterion of the test, and a purple color indicates that the ATP concentration is higher than the criterion. The method was applied to the assay of ATP extracted from Escherichia coli cells added to cow milk.

Keywords: Microbial contamination; ATP assay; Colorimetry; Enzymatic cycling; Xylenol orange; Escherichia coli

Classification of microheterogeneity in solid samples using µXRF by John L. Molloy; John R. Sieber (pp. 995-1001).

Micro X-ray fluorescence (µXRF) has been used nondestructively to investigate elemental heterogeneity by constructing two-dimensional maps of elemental concentrations in reference materials. µXRF probes sample sizes well below the 100 mg mass usually recommended for reference materials by NIST. Multivariate methods of analysis, such as principal-component analysis (PCA), show promise in identifying whether “nugget” effects exist within a material, where an element is enriched in small, isolated areas of the sample. The PCA model is built based on data taken in one location and compared with each elemental map. This methodology is shown for several reference materials including SRM 2702 and SRM 2703 to show how PCA treatment can be used to identify which elements exhibit nugget effects within the sub-mg mass range. A method of calculating the minimum recommended mass for solid samples is suggested using PCA iteratively on X-ray maps from which adjacent data points have been averaged. This is repeated until the mass sampled in a map is indistinguishable from data taken at a single location, suggesting no nugget effects can be detected. For SRM 1577c, a mass as low as 370 µg can be used without measurable nugget effects. Figure Microbeam x-ray fluorescence map showing heterogeneity present for Ti in SRM 2702 (Inorganics in Marine Sediment)

Keywords: µXRF; Microheterogeneity; PCA; Reference materials; Elemental analysis

Comparison of microemulsion electrokinetic chromatography with high-performance liquid chromatography for fingerprint analysis of resina draconis by Yuhua Cao; Wenjun Gong; Nan Li; Changna Yin; Yun Wang (pp. 1003-1010).

Microemulsion electrokinetic chromatography (MEEKC) has been developed for fingerprint analysis of resina draconis, a substitute for sanguis draconis in the Chinese market. The microemulsion as the running buffer was made up of 3.3% (w/v) sodium dodecyl sulfate (SDS), 6.6% (w/v) n-butanol, 0.8% (w/v) n-octane, and 10 mmol/L sodium tetraborate buffer (pH 9.2), which was also used as the solvent for ultrasonic extraction of both water- and fat-soluble compounds in the traditional Chinese medicine samples. Four batches of resina draconis obtained from different pharmaceutical factories located in different geographic regions were used to establish the electrophoretic fingerprint. MEEKC was performed using a Beckman PACE/MDQ system equipped with a diode-array detector and with monitoring at 280 nm. The fingerprint of resina draconis comprised 27 common peaks within 100 min. The relative standard deviations of the relative migration time of these common peaks were less than 2.1%. Through repetitive injection of the sample solution six times in 24 h, all relative standard deviations of the migration time and peak area of loureirin A and loureirin B were less than 2.5 and 3.8%, which demonstrated that the method had good stability and reproducibility. The relative peak areas of these common peaks in the electropherograms of four batches of resina draconis were processed with two mathematical methods, the correlation coefficient and the interangle cosine, to valuate the similarity. The values of the similarity degree of all samples were more than 0.91, which showed resina draconis samples from different origins were consistent. On the other hand, high-performance liquid chromatography (HPLC) coupled with photodiode-array detection was also applied to establish the fingerprint of resina draconis. The samples were separated with a LiChrospher C₁₈ column using acetonitrile (solvent A) and water containing 0.1% H₃PO₄ (solvent B) as the mobile phase in linear gradient elution mode at a flow rate of 0.6 mL/min and detection was at 280 nm. There were only 20 common peaks in the HPLC fingerprint, and the values of the similarity degree of all samples were also more than 0.91. Though the similarity results of fingerprint analysis seemed to be the same, MEEKC resulted in more common peaks and higher separation efficiency for a variety of polarities of the components than HPLC. So, MEEKC was more suitable for development of the fingerprint of resina draconis.

Keywords: Microemulsion electrokinetic chromatography; High-performance liquid chromatography; Resina draconis; Fingerprint

Rapid, single-step nucleic acid detection by Kyle A. Cissell; Sean Campbell; Sapna K. Deo (pp. 1011-1011).

2009 Bunsen-Kirchhoff Award for Analytical Spectroscopy (pp. 1013-1013).

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Analytical and Bioanalytical Chemistry (v.392, #5)

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Analytical and Bioanalytical Chemistry (v.392, #5)