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Analytical and Bioanalytical Chemistry (v.405, #1)

News from ABC: Editorial board and features by Steffen Pauly (pp. 1-2).

Happy New Year—renewal, welcome, and farewell by Cynthia K. Larive (pp. 3-5).

is Professor of Analytical Chemistry and chair of the Department of Chemistry at the University of California Riverside. Her research includes the development of microcoil NMR detectors for online electrophoretic separations, characterization of heparin and heparan sulfate, and the application of metabonomics and metabolic profiling to study hypoxia in plants and cancer. Dr. Larive also serves as editor-in-chief of the Analytical Sciences Digital Library, a free internet resource for educators, students, and practitioners.

Project-based learning: improving student engagement and performance in the laboratory by Jill K. Robinson (pp. 7-13).

is a senior lecturer in the Chemistry Department at Indiana University Bloomington, where she teaches general, environmental, and analytical chemistry courses. In recent years, she has implemented project-based learning at all levels, and curriculum reform efforts have been supported by several teaching-related grants. Her excellence in teaching has been recognized by the Board of Trustees Award and the President’s Award for Distinguished Teaching. She is also involved in outreach efforts to promote nanoscience and environmental education in middle and high schools throughout Indiana.

The copper pots and jam challenge by Hervé This (pp. 15-16).

Solution to peptide sequencing challenge by Juris Meija (pp. 17-17).

Fourth EuCheMS Chemistry Congress in Prague, Czech Republic by Sabrina Rau; Urs Hilbig (pp. 19-20).

Defining a molecular portrait of physical fitness by Adam Clouse; Sapna Deo; Evadnie Rampersaud; Jeff Farmer; Pascal J. Goldschmidt-Clermont; Sylvia Daunert (pp. 21-26).

is a graduate student advised by Dr. Sylvia Daunert at the University of Miami. His main research interests are in the area of bioanalytical chemistry. is an Associate Professor and Graduate Program Director of Biochemistry and Molecular Biology in the Miller School of Medicine at the University of Miami. She is also the Director of Molecular Medicine Pathway Program at the Miller School of Medicine. Her laboratory employs bionanotechnology principles in the development of sensing and drug delivery applications. Her group is involved in the development of novel bioanalytical techniques for the detection of biomolecules in situ, employing fluorescent and bioluminescent proteins. Sapna Deo is an editor of the book Photoproteins in Bioanalysis and also a recipient of the National Science Foundation Faculty CAREER Award. is a genetic epidemiologist with expertise in genomic studies of exercise responsiveness and obesity. She directs the Genetics of Exercise and Research (GEAR) study at the University of Miami, which aims to identify genes that show differential expression among individuals undergoing exercise training. is Program Development and Project Manager of the Genetics, Exercise and Research (GEAR) Program. He is responsible for providing leadership, direction, training and instruction, including managing day to day operational activities for the GEAR Program. J. Farmer has extensive experience in health and fitness management and well-established connections within the South Florida medical and business community. A lifelong exercise and fitness advocate, he has invested over 30 years in the health and fitness industry and has been actively involved in a wide variety of related activities, including a stint as a professional wrestler. is an internationally renowned cardiologist and cardiovascular researcher. He is Senior Vice President for Medical Affairs and Dean of the University of Miami Leonard M. Miller School of Medicine. He also serves as Chief Executive Officer of the University of Miami Health System (UHealth), which includes six hospitals and more than two dozen outpatient facilities. is the Lucille P. Markey Chair of Biochemistry and Molecular Biology at the Miller School of Medicine of the University of Miami. Her group employs recombinant DNA technology to design new molecular diagnostic tools and biosensors based on genetically engineered proteins and cells that find biomedical, environmental, and pharmaceutical applications. Additionally the research of the group focuses on the design of sensing arrays for the detection of molecules in small volumes and microfluidic platforms, and in the development of biomaterials for responsive drug delivery systems.

Isotopic labeling-assisted metabolomics using LC–MS by C. Bueschl; R. Krska; B. Kluger; R. Schuhmacher (pp. 27-33).

Metabolomics has emerged as the latest of the so-called “omics” disciplines and has great potential to provide deeper understanding of fundamental biochemical processes at the biological system level. Among recent technological developments, LC–HRMS enables determination of hundreds to thousands of metabolites over a wide range of concentrations and has developed into one of the most powerful techniques in non-targeted metabolomics. The analysis of mixtures of in-vivo-stable isotopic-labeled samples or reference substances with un-labeled samples leads to specific LC–MS data patterns which can be systematically exploited in practically all data-processing steps. This includes recognition of true metabolite-derived analytical features in highly complex LC–MS data and characterization of the global biochemical composition of biological samples. In addition, stable-isotopic labeling can be used for more accurate quantification (via internal standardization) and identification of compounds in different organisms.

Keywords: Bioanalytical methods; Mass spectrometry; Metabolomics; Liquid chromatography

Enhancement of biological mass spectrometry by using separations based on changes in ion mobility (FAIMS and DMS) by Randy W. Purves (pp. 35-42).

Analysis of complex biological samples for low-level analytes by liquid chromatography–tandem mass spectrometry (LC–MS/MS) often requires additional selectivity. Differential mobility techniques (FAIMS and DMS) have been shown to enhance LC–MS/MS analyses by separating ions in the gas-phase on a millisecond timescale by use of a mechanism that is complementary to both liquid chromatography and mass spectrometry. In this overview, a simplified description of the operation of these devices is given and an example presented that illustrates the utility of FAIMS (DMS) for solving a challenging analytical assay. Important recent advances in the field, including work with gas modifiers, are presented, along with an outlook for the technology. Figure FAIMS device for use with mass spectrometry

Keywords: Bioanalytical methods; Biological samples; HPLC; Mass spectrometry; FAIMS; DMS

Single-molecule monitoring in living cells by use of fluorescence microscopy by Wangxi Luo; Kangmin He; Tie Xia; Xiaohong Fang (pp. 43-49).

Monitoring single molecules in living cells is becoming a powerful tool for study of the location, dynamics, and kinetics of individual biomolecules in real time. In recent decades, several optical imaging techniques, for example epi-fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), confocal microscopy, quasi-TIRFM, and single-point edge excitation subdiffraction microscopy (SPEED), have been developed, and their capability of capturing single-molecule dynamics in living cells has been demonstrated. In this review, we briefly summarize recent advances in the use of these imaging techniques for monitoring single-molecules in living cells for a better understanding of important biological processes, and discuss future developments.

Keywords: Single-molecule imaging; Fluorescence microscopy; TIRFM; Living cell

Towards analytically useful two-dimensional Fourier transform ion cyclotron resonance mass spectrometry by Maria A. van Agthoven; Marc-André Delsuc; Geoffrey Bodenhausen; Christian Rolando (pp. 51-61).

Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) achieves high resolution and mass accuracy, allowing the identification of the raw chemical formulae of ions in complex samples. Using ion isolation and fragmentation (MS/MS), we can obtain more structural information, but MS/MS is time- and sample-consuming because each ion must be isolated before fragmentation. In 1987, Pfändler et al. proposed an experiment for 2D FT-ICR MS in order to fragment ions without isolating them and to visualize the fragmentations of complex samples in a single 2D mass spectrum, like 2D NMR spectroscopy. Because of limitations of electronics and computers, few studies have been conducted with this technique. The improvement of modern computers and the use of digital electronics for FT-ICR hardware now make it possible to acquire 2D mass spectra over a broad mass range. The original experiments used in-cell collision-induced dissociation, which caused a loss of resolution. Gas-free fragmentation modes such as infrared multiphoton dissociation and electron capture dissociation allow one to measure high-resolution 2D mass spectra. Consequently, there is renewed interest to develop 2D FT-ICR MS into an efficient analytical method. Improvements introduced in 2D NMR spectroscopy can also be transposed to 2D FT-ICR MS. We describe the history of 2D FT-ICR MS, introduce recent improvements, and present analytical applications to map the fragmentation of peptides. Finally, we provide a glossary which defines a few keywords for the 2D FT-ICR MS field.

Keywords: Mass spectrometry; Fourier transform ion cyclotron resonance; Two-dimensional; FT-ICR

Advances in pesticide biosensors: current status, challenges, and future perspectives by Shaoqin Liu; Zhaozhu Zheng; Xinyu Li (pp. 63-90).

Public concern over pesticide residues has been increasing dramatically owing to the high toxicity and bioaccumulation effects of pesticides and the serious risks that they pose to the environment and human health. It is therefore crucial to monitor pesticide residues by using various analytical methods and techniques, especially highly sensitive, highly selective, simple, rapid, cost-effective, and portable ones. Biosensor strategies have become research hotspots and ideal candidates for pesticide detection, having such features as high sensitivity, fast response, robustness, low cost and miniaturization, as well as in situ and real-time monitoring. This review covers advances in the design and fabrication of biosensors for pesticide detection since 2005. Special emphasis is placed on the state-of-art selection of receptors, the use of different transduction techniques and fast screening strategies, and the application of various biosensors developed in food and environmental safety. Both advantages and drawbacks of these techniques are then summarized. Finally, challenges, strategies, and perspectives in further developing pesticide biosensors are also discussed. Figure Principle of operation of pesticide biosensors

Keywords: Pesticide residue detection; Electrochemical biosensor; Optical biosensor; Enzyme biosensor; Immunosensor

Application of quantitative PCR for the detection of microorganisms in water by Marelize Botes; Michéle de Kwaadsteniet; Thomas Eugene Cloete (pp. 91-108).

The occurrence of microorganisms in water due to contamination is a health risk and control thereof is a necessity. Conventional detection methods may be misleading and do not provide rapid results allowing for immediate action. The quantitative polymerase chain reaction (qPCR) method has proven to be an effective tool to detect and quantify microorganisms in water within a few hours. Quantitative PCR assays have recently been developed for the detection of specific adeno- and polyomaviruses, bacteria and protozoa in different water sources. The technique is highly sensitive and able to detect low numbers of microorganisms. Quantitative PCR can be applied for microbial source tracking in water sources, to determine the efficiency of water and wastewater treatment plants and act as a tool for risk assessment. Different qPCR assays exist depending on whether an internal control is used or whether measurements are taken at the end of the PCR reaction (end-point qPCR) or in the exponential phase (real-time qPCR). Fluorescent probes are used in the PCR reaction to hybridise within the target sequence to generate a signal and, together with specialised systems, quantify the amount of PCR product. Quantitative reverse transcription polymerase chain reaction (q-RT-PCR) is a more sensitive technique that detects low copy number RNA and can be applied to detect, e.g. enteric viruses and viable microorganisms in water, and measure specific gene expression. There is, however, a need to standardise qPCR protocols if this technique is to be used as an analytical diagnostic tool for routine monitoring. This review focuses on the application of qPCR in the detection of microorganisms in water.

Keywords: Water; Waste/sludge; Quality assurance/control; PCR

Molecular characterization of dissolved organic matter (DOM): a critical review by Antonio Nebbioso; Alessandro Piccolo (pp. 109-124).

Advances in water chemistry in the last decade have improved our knowledge about the genesis, composition, and structure of dissolved organic matter, and its effect on the environment. Improvements in analytical technology, for example Fourier-transform ion cyclotron (FT-ICR) mass spectrometry (MS), homo and hetero-correlated multidimensional nuclear magnetic resonance (NMR) spectroscopy, and excitation emission matrix fluorimetry (EEMF) with parallel factor (PARAFAC) analysis for UV–fluorescence spectroscopy have resulted in these advances. Improved purification methods, for example ultrafiltration and reverse osmosis, have enabled facile desalting and concentration of freshly collected DOM samples, thereby complementing the analytical process. Although its molecular weight (MW) remains undefined, DOM is described as a complex mixture of low-MW substances and larger-MW biomolecules, for example proteins, polysaccharides, and exocellular macromolecules. There is a general consensus that marine DOM originates from terrestrial and marine sources. A combination of diagenetic and microbial processes contributes to its origin, resulting in refractory organic matter which acts as carbon sink in the ocean. Ocean DOM is derived partially from humified products of plants decay dissolved in fresh water and transported to the ocean, and partially from proteinaceous and polysaccharide material from phytoplankton metabolism, which undergoes in-situ microbial processes, becoming refractory. Some of the DOM interacts with radiation and is, therefore, defined as chromophoric DOM (CDOM). CDOM is classified as terrestrial, marine, anthropogenic, or mixed, depending on its origin. Terrestrial CDOM reaches the oceans via estuaries, whereas autochthonous CDOM is formed in sea water by microbial activity; anthropogenic CDOM is a result of human activity. CDOM also affects the quality of water, by shielding it from solar radiation, and constitutes a carbon sink pool. Evidence in support of the hypothesis that part of marine DOM is of terrestrial origin, being the result of a long-term carbon sedimentation, has been obtained from several studies discussed herein.

Keywords: Dissolved organic matter; Carbon cycle; Humic substances; Water; Dissolved organic carbon; Natural products

Microextraction techniques combined with capillary electrophoresis in bioanalysis by Isabelle Kohler; Julie Schappler; Serge Rudaz (pp. 125-141).

Over the past two decades, many environmentally sustainable sample-preparation techniques have been proposed, with the objective of reducing the use of toxic organic solvents or substituting these with environmentally friendly alternatives. Microextraction techniques (MEs), in which only a small amount of organic solvent is used, have several advantages, including reduced sample volume, analysis time, and operating costs. Thus, MEs are well adapted in bioanalysis, in which sample preparation is mandatory because of the complexity of a sample that is available in small quantities (mL or even μL only). Capillary electrophoresis (CE) is a powerful and efficient separation technique in which no organic solvents are required for analysis. Combination of CE with MEs is regarded as a very attractive environmentally sustainable analytical tool, and numerous applications have been reported over the last few decades for bioanalysis of low-molecular-weight compounds or for peptide analysis. In this paper we review the use of MEs combined with CE in bioanalysis. The review is divided into two sections: liquid and solid-based MEs. A brief practical and theoretical description of each ME is given, and the techniques are illustrated by relevant applications.

Keywords: Bioanalysis; Capillary electrophoresis; Environmentally sustainable chemistry; Green chemistry; Microextraction; Sample preparation

Determination of perfluorinated compounds in aquatic organisms: a review by Sara Valsecchi; Marianna Rusconi; Stefano Polesello (pp. 143-157).

Bioaccumulation of PFAS in aquatic organisms is an environmental problem of growing concern around the world. This problem has been tackled by regulatory bodies by proposing EQS for biota in EU water bodies and tolerable daily intake for food. The introduction of regulatory limits requires the availability of harmonised and validated analytical methods of sufficient sensitivity. This paper reviews recent advances in analytical methods for analysis of PFAS in aquatic organisms. The methods available for biota analysis are mostly based on three extraction procedures: ion-pair extraction, solvent liquid extraction, and alkaline digestion. The resulting extracts are then subjected to different clean-up or enrichment steps on solid adsorbents, for example graphitized carbon black, C₁₈, and WAX phases. All methods reviewed in this work give reliable results but are partially validated only, because of the lack of certified reference materials and regular interlaboratory exercises. The few interlaboratory exercises performed on real unspiked samples did not afford satisfactory results for PFAS other than PFOS, especially for matrices with high lipid content, for example mussels. The reasons for those partially negative results have been identified, and can mainly be attributed to calibration procedures and availability and purity of standards. The urgent need for certified reference materials for this type of analysis is emphasized.

Keywords: Perfluorinated compounds; Extraction methods; LC–MS; Aquatic organisms; Review

δ ¹⁵N measurement of organic and inorganic substances by EA-IRMS: a speciation-dependent procedure by Natacha Gentile; Michel J. Rossi; Olivier Delémont; Rolf T. W. Siegwolf (pp. 159-176).

Little attention has been paid so far to the influence of the chemical nature of the substance when measuring δ ¹⁵N by elemental analysis (EA)–isotope ratio mass spectrometry (IRMS). Although the bulk nitrogen isotope analysis of organic material is not to be questioned, literature from different disciplines using IRMS provides hints that the quantitative conversion of nitrate into nitrogen presents difficulties. We observed abnormal series of δ ¹⁵N values of laboratory standards and nitrates. These unexpected results were shown to be related to the tailing of the nitrogen peak of nitrate-containing compounds. A series of experiments were set up to investigate the cause of this phenomenon, using ammonium nitrate (NH₄NO₃) and potassium nitrate (KNO₃) samples, two organic laboratory standards as well as the international secondary reference materials IAEA-N1, IAEA-N2—two ammonium sulphates [(NH₄)₂SO₄]—and IAEA-NO-3, a potassium nitrate. In experiment 1, we used graphite and vanadium pentoxide (V₂O₅) as additives to observe if they could enhance the decomposition (combustion) of nitrates. In experiment 2, we tested another elemental analyser configuration including an additional section of reduced copper in order to see whether or not the tailing could originate from an incomplete reduction process. Finally, we modified several parameters of the method and observed their influence on the peak shape, δ ¹⁵N value and nitrogen content in weight percent of nitrogen of the target substances. We found the best results using mere thermal decomposition in helium, under exclusion of any oxygen. We show that the analytical procedure used for organic samples should not be used for nitrates because of their different chemical nature. We present the best performance given one set of sample introduction parameters for the analysis of nitrates, as well as for the ammonium sulphate IAEA-N1 and IAEA-N2 reference materials. We discuss these results considering the thermochemistry of the substances and the analytical technique itself. The results emphasise the difference in chemical nature of inorganic and organic samples, which necessarily involves distinct thermochemistry when analysed by EA-IRMS. Therefore, they should not be processed using the same analytical procedure. This clearly impacts on the way international secondary reference materials should be used for the calibration of organic laboratory standards. Figure Control chart of the δ ¹⁵N value of IAEA-N1, IAEA-NO-3 and NH₄NO₃ analysed a) with oxygen injection (analytical cycle 70 s, oxygen for 60 s, sample start and stop at 18 s/20 s), b) with oxygen injection (analytical cycle 70 s, oxygen for 60 s, sample start and stop at 0 s/2 s and 5 s/7 s), c) without oxygen injection (analytical cycle 70 s, sample start and stop at 18 s/20 s).

Keywords: Nitrate; Nitrogen; Isotope ratio mass spectrometry; Ammonium; Elemental analysis–isotope ratio mass spectrometry; Inorganic

Negative ion tandem mass spectrometry of prenylated fungal metabolites and their derivatives by Ramona Heinke; Norbert Arnold; Ludger Wessjohann; Jürgen Schmidt (pp. 177-189).

Liquid chromatography negative ion electrospray ionisation tandem mass spectrometry has been used for characterisation of naturally occurring prenylated fungal metabolites and synthetic derivatives. The fragmentation studies allow an elucidation of the decomposition pathways for these compounds. It could be shown, that the prenyl side chain is degraded by successive radical losses of C₅ units. Both the benzoquinones and the phenolic derivatives display significant key ions comprising the aromatic ring. In some cases, the formation of significant oxygen-free key ions could be evidenced by high-resolution MS/MS measurements. Furthermore, the different types of basic skeletons, benzoquinones and phenol type as well as cyclic prenylated compounds, can be differentiated by their MS/MS behaviour. Figure Fruiting bodies of Suillus bovinus, the structure of boviquinone-4 and its negative ion MS2 spectrum

Keywords: Meroterpenoids; Prenylated fungal metabolites; Boviquinones; Fungi; Basidiomycetes; Negative ion ESI-MS/MS

Mass spectrometric profiling of Bacillus cereus strains and quantitation of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2 bioassay by Timo Stark; Sandra Marxen; Andrea Rütschle; Genia Lücking; Siegfried Scherer; Monika Ehling-Schulz; Thomas Hofmann (pp. 191-201).

A fast and robust high-throughput ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC–TOF MS) profiling method was developed and successfully applied to discriminate a total of 78 Bacillus cereus strains into no/low, medium and high producers of the emetic toxin cereulide. The data obtained by UPLC–TOF MS profiling were confirmed by absolute quantitation of cereulide in selected samples by means of high-performance liquid chromatography with tandem mass spectrometry (HPLC–MS/MS) and stable isotope dilution assay (SIDA). Interestingly, the B. cereus strains isolated from four vomit samples and five faeces samples from patients showing symptoms of intoxication were among the group of medium or high producers. Comparison of HEp-2 bioassay data with those determined by means of mass spectrometry showed differences, most likely because the HEp-2 bioassay is based on the toxic action of cereulide towards mitochondria of eukaryotic cells rather than on a direct measurement of the toxin. In conclusion, the UPLC–electrospray ionization (ESI)–TOF MS and the HPLC–ESI–MS/MS–SIDA analyses seem to be promising tools for the robust high-throughput analysis of cereulide in B. cereus cultures, foods and other biological samples. Figure Score plot (comp[1] vs. comp[2]) of UPLC‐TOF MS full scan analysis (50–1,300 Da) of 78 B. cereus strains with color‐coded signal intensity of the accurate mass of pseudo molecular ion of cereulide (m/z 1175.6608, [M+Na]⁺), from group 1 with the lowest up to group 5 with the highest signal intensity

Keywords: Cereulide; Stable isotope dilution analysis; ¹³C₆ cereulide; Bacillus cereus ; HEp-2 assay

A single run LC-MS/MS method for phospholipidomics by Corinne Buré; Sophie Ayciriex; Eric Testet; Jean-Marie Schmitter (pp. 203-213).

Liquid chromatography coupled to tandem mass spectrometry has been compared to shotgun analysis with the objective of finding the best compromise for a single run analysis of whole cell phospholipids. Hydrophilic interaction liquid chromatography (HILIC), normal phase (NP), and reversed phase (RP) liquid chromatography were evaluated with reference phospholipids belonging to phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) classes. NP-HPLC- and RP-HPLC-ESI-MS/MS were applied to yeast phospholipidome analysis, using a wild-type strain and two strains defective for acyltransferases that are known to be involved in de novo phospholipid synthesis or phospholipid remodeling. The MRM mode was used for relative quantitation of individual compounds based on reference phospholipids bearing fatty acid chains with an odd number of carbon atoms. Combined LC-MS/MS was found superior to shotgun analysis, leading to a larger number of quantified species than shotgun analysis. Finally, RP-HPLC-MS/MS was the preferred method for its higher selectivity, robustness, and better repeatability.

Keywords: Liquid chromatography; Shotgun; Mass spectrometry; Phospholipids

Validation of an LC-MS/MS method for the quantitative determination of mavoglurant (AFQ056) in human plasma by Annamaria Jakab; Serge Winter; Marc Raccuglia; Frank Picard; Swati Dumitras; Ralph Woessner; Sanket Mistry; Jayraj Chudasama; Swati Guttikar; Olivier Kretz (pp. 215-223).

A simple, sensitive, and selective liquid chromatography/tandem mass spectrometry method was validated for the identification and quantification of mavoglurant (AFQ056) in human plasma. The chromatographic separation was performed using a Cosmosil 5 C18 (150 × 4.6 mm, 5 μm) column at 40 ± 0.5 °C with a mobile phase consisting of acetic acid in water (0.1 %, v/v)/methanol (10:90, v/v) with a flow rate of 1.0 mL/min followed by quantification with tandem mass spectrometry, operating with electrospray ionization in positive ion mode and applying multiple reaction monitoring. The validated method described in this paper presents high absolute recovery with precision and accuracy meeting the acceptance criteria. The method was precise and accurate for 2- and 10-fold dilution of samples. The method was validated using sodium heparin as specific anticoagulant, and the anticoagulant effect was tested by lithium heparin and K₃EDTA. The method was successfully cross-validated between two bioanalytical sites. The method was specific for mavoglurant within the given criteria for acceptance (apparent peak area at the retention time of mavoglurant in zero samples was less than 20 % compared with the mean peak area at LLOQ) in human plasma. The method was fully validated for the quantitative determination of mavoglurant in human plasma between the range of 2.00 and 2,500 ng/mL.

Keywords: AFQ056; Mavoglurant; mGluR5; LC-MS/MS; Human plasma; Recovery; Validation

Advantages and limitations of a new cationic coating inducing a slow electroosmotic flow for CE-MS peptide analysis: a comparative study with commercial coatings by Martin Pattky; Carolin Huhn (pp. 225-237).

Capillary coatings are crucial for high-quality separation performance in capillary electrophoresis analysis of proteins or peptides as they prevent analyte adsorption at the capillary wall. These coating materials have to fulfill many requirements such as a good separation performance and ensuring a good repeatability. The number of commercially available coating materials is still limited, especially with regard to the charge density on the coating material and the induced electroosmotic flow (EOF) velocity. In this work, we compare the separation performance of the novel self-made cationic capillary coating OHNOON and two commercially available coating materials, the acrylamide based, neutral LN® and the cationic hexadimethrine bromide (Polybrene), using the same coating procedure for all three coating materials. The coatings are investigated regarding the separation efficiency, analyte resolution, coating stability, and migration time stability in tryptic peptide analysis. Good separation performance was confirmed for all three coating materials: all coatings provided high plate numbers of up to 400,000–500,000 and a repeatability of the EOF and the analyte migration times in the range of 1 % relative standard deviation or below. Our results reveal a moderate EOF velocity for the novel OHNOON coating in comparison to the Polybrene coating. We present a detailed discussion of the impact of this reduced EOF velocity and the separation performance. The results presented here will help to define the necessary properties of coating materials to achieve the best compromise between speed of analysis and resolution for the respective application. We show that our novel OHNOON coating is especially valuable for the analysis of low mobility analytes and for samples with a broad range of analyte mobilities.

Keywords: Cationic adsorbed coating; Polybrene; Separation optimization; EOF variation; OHNOON

An automated method for measurement of methoxetamine in human plasma by use of turbulent flow on-line extraction coupled with liquid chromatography and mass spectrometric detection by Emuri Abe; Florian Ricard; François Darrouzain; Jean Claude Alvarez (pp. 239-245).

Methoxetamine is a new ketamine derivative designer drug which has recently become available via the Internet marketed as “legal ketamine”. It is a new dissociative recreational drug, acting as an NMDA receptor antagonist and dopamine reuptake inhibitor. The objective of this study was to develop on-line automated sample preparation using a TurboFlow device coupled with liquid chromatography with ion-trap mass spectrometric detection for measurement of methoxetamine in human plasma. Samples (100 μL) were vortex mixed with internal standard solution (ketamine-d4 in acetonitrile). After centrifugation, 20 μL of the supernatant was injected on to a 50 mm × 0.5-mm C18XL Turboflow column. The retained analytes were then back-flushed on to a 50 mm × 3-mm (3 μm) Hypersil Gold analytical column for chromatographic separation, then eluted with a formate buffer–acetonitrile gradient. Methoxetamine and the IS were ionized by electrospray in positive mode. Parent [M + H]⁺ ions were m/z 248.1 for methoxetamine and m/z 242.0 for the IS. The most intense product ions from methoxetamine (m/z 203.0) and the IS (m/z 224.0) were used for quantification. The assay was accurate (96.8–108.8 % range) and precise (intra and inter-day coefficients of variation <8.8 %) over the range of 2.0 (lower limit of quantification) to 1000.0 ng mL⁻¹ (upper limit of quantification). No matrix effect was observed. This method has been successfully applied to determination of plasma concentrations of methoxetamine in the first French hospitalization case report after acute intoxication; the plasma concentration was 136 ng mL⁻¹.

Keywords: Designer drugs; Methoxetamine; On-line extraction; LC–MS–MS

Development and validation of two LC-MS/MS methods for the detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine using turbulent flow chromatography by Nadine Schaefer; Benjamin Peters; Peter Schmidt; Andreas H. Ewald (pp. 247-258).

In the context of driving ability diagnostics in Germany, administrative cutoffs for various drugs and pharmaceuticals in urine have been established. Two liquid chromatography–tandem mass spectrometry methods for simultaneous detection and quantification of amphetamines, designer amphetamines, benzoylecgonine, benzodiazepines, opiates, and opioids in urine were developed and validated. A 500-μL aliquot of urine was diluted and fortified with an internal standard solution. After enzymatic cleavage, online extraction was performed by an ion-exchange/reversed-phase turbulent flow column. Separation was achieved by using a reversed-phase column and gradient elution. For detection, a Thermo Fisher TSQ Quantum Ultra Accurate Mass tandem mass spectrometer with positive electrospray ionization was used, and the analytes were measured in multiple-reaction monitoring mode detecting two transitions per precursor ion. The total run time for both methods was about 15 min. Validation was performed according to the guidelines of the Society of Toxicological and Forensic Chemistry. The results of matrix effect determination were between 78 % and 116 %. The limits of detection and quantification for all drugs, except zopiclone, were less than10 ng/mL and less than 25 ng/mL, respectively. Calibration curves ranged from 25 to 200 ng/mL for amphetamines, designer amphetamines, and benzoylecgonine, from 25 to 250 ng/mL for benzodiazepines, from 12.5 to 100 ng/mL for morphine, codeine, and dihydrocodeine, and from 5 to 50 ng/mL for buprenorphine and norbuprenorphine. Intraday and interday precision values were lower than 15 %, and bias values within ± 15 % were achieved. Turbulent flow chromatography needs no laborious sample preparation, so the workup is less time-consuming compared with gas chromatography–mass spectrometry methods. The methods are suitable for quantification of multiple analytes at the cutoff concentrations required for driving ability diagnostics in Germany.

Keywords: Turbulent flow chromatography; Liquid chromatography-tandem mass spectrometry; Driving ability diagnostics; Validation

Analysis of carbamazepine and its five metabolites in serum by large-volume sample stacking–sweeping capillary electrophoresis by Yan-Yu Lin; Chun-Chi Wang; Yu-Hsiang Ho; Cheng-Sheng Chen; Shou-Mei Wu (pp. 259-266).

This study establishes a method, using different buffer conductivities and large-volume sample stacking (LVSS)–sweeping capillary electrophoresis, for analysis of carbamazepine (CBZ) and its five metabolites in serum. The capillary (50/60 cm) was filled with a high concentration of background electrolyte (150 mM phosphate, pH 3.5, containing 15 % methanol), followed by a large volume of samples (10 psi, 20 s) with low-concentration buffers (5 mM phosphate, pH 3.5, with 5 % methanol). When high voltage was applied (−20 kV), the sodium dodecyl sulfate (SDS) started to sweep the analytes to an outlet. Meanwhile, the analytes decelerated at the boundary between low- and high-conductivity buffers. Finally, a narrow sample zone was formed. The procedure of sweeping and separation was simultaneously carried out by a sweeping buffer (150 mM phosphate, pH 3.5) with 15 % methanol and 50 mM SDS added, and the detection was performed by UV at 214 nm. The method was validated for linearity (r ≧ 0.997), precision, and accuracy. The calibration curves were established for CBZ and its five metabolites between 0.03–25 and 0.03–3 μg/mL. The limits of detection (S/N = 3) were 0.01 μg/mL for each analyte. Compared with simple MEKC (0.5 psi, 5 s), this system can improve the sensitivity about 300-fold. Finally, this method was successfully applied to five patients, who had taken 200 mg CBZ daily, and CBZ levels were found to be from 3.72 to 5.82 μg/mL. Figure Chromatogram of resolution of analytes extracted from serum by LVSS-sweeping CE.; peaks: 1. CBZ, 2. CM-3, 3. CM-E, 4. CM-2, 5. CM-10, 6. CM-D, IS: ethyl paraben

Keywords: Capillary electrophoresis; Carbamazepine; Metabolites; Serum; LVSS-sweeping

Enantioselective analysis of zopiclone in rat brain by liquid chromatography tandem mass spectrometry by Milena Araújo Tonon; Valquíria A. P. Jabor; Pierina Sueli Bonato (pp. 267-273).

A new high-performance liquid chromatographic method with triple quadrupole mass spectrometry detection was developed and validated for the quantification of zopiclone enantiomers in rat brain samples. Zopiclone enantiomers were resolved on a CHIRALPAK AD column with a mobile phase consisting of acetonitrile/ethanol/methanol (60:20:20, v/v/v) at a flow rate of 1.3 mL min^-1. Moclobemide was used as internal standard. The sample treatment procedure was carried out employing solid-phase extraction, yielding mean absolute recoveries of 89.6 and 91.7 % for each zopiclone enantiomer. The validated method showed linearity in the range of 0.29–344.8 ng g⁻¹, with quantification limits of 0.29 ng g⁻¹ for both enantiomers. Precision and accuracy were within acceptable levels of confidence (<15 %). The method was applied in a pilot study of zopiclone kinetic disposition in rats. It could be observed that the levels of (+)-(S)-zopiclone were always higher than those of (-)-(R)-zopiclone, confirming the stereoselective disposition of zopiclone.

Keywords: Zopiclone; LC-MS-MS; Solid-phase extraction; Chiral kinetic disposition; Brain

Covalent attachment of functionalized cardiolipin on a biosensor gold surface allows repetitive measurements of anticardiolipin antibodies in serum by Alice Schlichtiger; Claudia Baier; Meng-Xin Yin; Andrew B. Holmes; Makiko Maruyama; Ralf Strasser; Ulrich Rant; Markus Thaler; Peter B. Luppa (pp. 275-285).

Antiphospholipid antibodies (aPL) are a relevant serological indicator of antiphospholipid syndrome (APS). A solid-state surface with covalently bound ω-amine-functionalized cardiolipin was established and the binding of β2-glycoprotein I (β2-GPI) was investigated either by use of surface plasmon resonance (SPR) biosensor, by electrically switchable DNA interfaces (switchSENSE) and by scanning tunneling microscopy (STM). STM could clearly visualize the attachment of β2-GPI to the cardiolipin surface. Using the switchSENSE sensor, β2-GPI as specific ligand could be identified by increased hydrodynamic friction. The binding of anti-cardiolipin antibodies (aCL) was detected against the ω-amine-functionalized cardiolipin-modified SPR biosensor (aCL biosensor) using sera from healthy donors, APS patients and syphilis patients. Our results showed that the aCL biosensor is a much more sensitive diagnostic device for APS patients compared to previous methods. The specificity between β2-GPI-dependent autoimmune- and β2-GPI-independent infection-associated types of aPLs was also studied and they can be distinguished by the different binding kinetics and patterns.

Keywords: Antiphospholipid syndrome; Antiphospholipid antibody; Cardiolipin; β2-glycoprotein I; Phospholipid; Surface plasmon resonance

Potentiometric propranolol-selective sensor based on molecularly imprinted polymer by O. Gurtova; L. Ye; F. Chmilenko (pp. 287-295).

A novel potentiometric sensor based on molecularly imprinted polymer (MIP) for propranolol, an adrenergic-blocking drug, was designed. The influence of molecularly imprinted polymer particle content and sodium tetraphenylborate additives in polyvinylchloride membrane was shown. The electrodes show near-Nernstian responses down to 10⁻⁴–10⁻⁵ M propranolol concentration. The potentiometric response of MIP-based sensor for propranolol in mixed nonaqueous medium was shown at first. Sensor selectivity relative to various inorganic cations, atenolol and metoprolol, was reported. Direct potentiometry was used to determine propranolol in aqueous modeling solutions and pharmaceutical preparations with good results.

Keywords: Molecular imprinted polymer; Propranolol; Potentiometric sensor; Nanoparticles; Anaprilin

A novel, disposable, screen-printed amperometric biosensor for ketone 3-β-hydroxybutyrate fabricated using a 3-β-hydroxybutyrate dehydrogenase–mesoporous silica conjugate by Takeshi Shimomura; Touru Sumiya; Masatoshi Ono; Tetsuji Ito; Taka-aki Hanaoka (pp. 297-305).

A disposable amperometric biosensor for ketone 3-β-hydroxybutyrate (3HB) has been developed successfully. The sensor is based on a screen-printed carbon electrode containing Meldola’s Blue (MB) and sensing components containing nicotinamide adenine dinucleotide (NAD⁺) and 3-β-hydroxybutyrate dehydrogenase (3HBDH) immobilized in mesoporous silica (FSM8.0) using an aqueous photo-cross-linkable polymer matrix of polyvinyl alcohol (O-391), and it requires only a small sample volume of 10 μL for the measurement. The behavior of a resulting biosensor, i.e., 3HBDH–FSM8.0/NAD⁺/MB-SPCE, was examined in terms of NAD⁺ concentration for construction, pH, applied potential, operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.6 and at an applied potential of −50 mV. The determination range and the response time for 3HB were from 30 μM to 8 mM and approximately 30 s, respectively. In addition, the sensor was quite stable and maintained >90 % of its initial response after being stored for over 6 months. This result implies that our method provides a novel biosensor for ketone 3-β-hydroxybutyrate which is easy-to-use, cost-effective, and has good reproducibility, which are vital for commercial purposes. Figure Layer structure and operation mechanism of 3HB biosensor

Keywords: Screen-printed carbon electrode; Meldola’s Blue; Nicotinamide adenine dinucleotide; 3-β-Hydroxybutyrate dehydrogenase; Mesoporous silica; Aqueous photo-cross-linkable polymer

Analysis of intercellular communication by flexible hydrodynamic gating on a microfluidic chip by Peng Chen; Pu Chen; Xiaojun Feng; Wei Du; Bi-Feng Liu (pp. 307-314).

Intercellular Ca²⁺ waves are propagation of Ca²⁺ transients among cells that could be initiated by chemical stimulation. Current methods for analyzing intercellular Ca²⁺ waves are difficult to realize localized chemical stimulations upon the target cell without interfering with adjacent contacting cells. In this paper, a simple and flexible microfluidic method was developed for investigating the intercellular communication of Ca²⁺ signals. A cross-patterned microfluidic chip was designed and fabricated with polydimethylsiloxane as the structural material. Localized chemical stimulation was achieved by a new strategy based on hydrodynamic gating technique. Clusters of target cells were seeded at the location within 300 μm downstream of the intersection of the cross-shaped microchannel. Confined lateral molecular diffusion largely minimized the interference from diffusion-induced stimulation of adjacent cells. Localized stimulation of the target cell with adenosine 5′-triphosphate successfully induced the propagation of intercellular Ca²⁺ waves among a population of adjacent contacting cells. Further inhibition studies verified that the propagation of calcium signals among NIH-3 T3 cells was dependent on direct cytosolic transfer via gap junctions. The developed microfluidic method provides a versatile platform for investigating the dynamics of intercellular communications. Fig Analysis of intercellular communication by flexible hydrodynamic gating

Keywords: Microfluidic chip; Hydrodynamic gating; Intercellular communication; Ca²⁺ waves

Characterization of in vitro transcription amplification linearity and variability in the low copy number regime using External RNA Control Consortium (ERCC) spike-ins by Jason G. Kralj; Marc L. Salit (pp. 315-320).

Using spike-in controls designed to mimic mammalian mRNA species, we used the quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the performance of in vitro transcription (IVT) amplification process of small samples. We focused especially on the confidence of the transcript level measurement, which is essential for differential gene expression analyses. IVT reproduced gene expression profiles down to approximately 100 absolute input copies. However, a RT-qPCR analysis of the antisense RNA showed a systematic bias against low copy number transcripts, regardless of sequence. Experiments also showed that noise increases with decreasing copy number. First-round IVT preserved the gene expression information within a sample down to the 100 copy level, regardless of total input sample amount. However, the amplification was nonlinear under low total RNA input/long IVT conditions. Variability of the amplification increased predictably with decreasing input copy number. For the small enrichments of interest in typical differential gene expression studies (e.g., twofold changes), the bias from IVT reactions is unlikely to affect the results. In limited cases, some transcript-specific differential gene expression values will need adjustment to reflect this bias. Proper experimental design with reasonable detection limits will yield differential gene expression capability even between low copy number transcripts.

Keywords: In vitro transcription; ERCC; RNA amplification; Amplification bias

Chemico-physical characterisation and in vivo biocompatibility assessment of DLC-coated coronary stents by Micaela Castellino; Vlad Stolojan; Alessandro Virga; Massimo Rovere; Karine Cabiale; Marco R. Galloni; Alberto Tagliaferro (pp. 321-329).

The vast majority of stent thrombosis occurs in the acute and sub-acute phases and is more common in patients with acute coronary syndromes, due to the thrombotic milieu where stent struts are positioned. Stent thrombosis is likely due to incomplete tissue coverage of metallic stents as the contact between metallic stents and blood elements may lead to platelet adhesion and trigger vessel thrombosis. If a stent is covered after 7 days, the risk that it will be found uncovered at later stages is very low (<1 %). In this article, we demonstrate that diamond-like carbon (DLC) coatings, deposited by physical vapour deposition, promote rapid endothelisation of coronary stent devices, with very low platelets activation, reducing thrombotic clots. We relate these behaviours to the surface and bulk material properties of the DLC films, subjected to a comprehensive chemico-physical characterisation using several techniques (X-ray photoelectron spectroscopy, atomic force microscopy, field-emission scanning electron microscope, transmission electron microscopy combined with electron energy loss spectroscopy, Raman and dispersive X-ray spectroscopy). In vivo studies, conducted on 24 pigs, have shown complete endothelisation after 7 days, with no fibrin mesh and with only rare monocytes scattered on the endothelial layer while 30 and 180 days tests have shown reduced inflammatory activation and a complete stabilisation of the vessel healing, with a minimal neointimal proliferation. The integral and permanent DLC film coating improves haemo- and bio-compatibility and leads to an excellent early vessel healing of the stent whilst the extremely thin strut thickness reduces the amount of late neointima and consequently the risk of late restenosis. These data should translate into a reduced acute and sub-acute stent thrombosis. Fig Carbon film-coated stent (SEM-×500 magnification). Detail of the endothelial layer.

Keywords: Biomaterials; Interface/surface analysis; Thin films; X-ray spectroscopy (XPS/XRF/EDX); Atomic force microscopy (AFM); Electron/ion microprobe

Simultaneous determination of testosterone, cortisol, and dehydroepiandrosterone in saliva by stable isotope dilution on-line in-tube solid-phase microextraction coupled with liquid chromatography–tandem mass spectrometry by Hiroyuki Kataoka; Kentaro Ehara; Rie Yasuhara; Keita Saito (pp. 331-340).

We have developed a simple and sensitive method for the simultaneous determination of testosterone (TES), cortisol (CRT), and dehydroepiandrosterone (DHEA) in saliva by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) using a Discovery HS F5 column. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 μL of sample at a flow rate of 200 μL/min using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. The in-tube SPME LC–MS/MS method showed good linearity with correlation coefficients r ≥ 0.9998 for TES, CRT, and DHEA using their respective stable isotope-labeled internal standards. The intra-day and inter-day precisions (relative standard deviations) were below 4.9 and 8.5 % (n = 5), respectively. This method was successfully utilized to analyze TES, CRT, and DHEA in saliva samples without any other pretreatment or interference peaks, and the quantification limits (S/N = 10) of TES, CRT and DHEA were about 0.01, 0.03 and 0.29 ng/mL saliva, respectively. The recoveries of these compounds spiked into saliva samples were each above 94 %. This method was applied to analyze changes in salivary TES, CRT, and DHEA levels resulting from stress and fatigue load.

Keywords: Testosterone; Cortisol; Dehydroepiandrosterone; Saliva; In-tube solid-phase microextraction; Liquid chromatography–tandem mass spectrometry

Determination of t,t-muconic acid in urine samples using a molecular imprinted polymer combined with simultaneous ethyl chloroformate derivatization and pre-concentration by dispersive liquid–liquid microextraction by Mohana Krishna Reddy Mudiam; Abhishek Chauhan; Krishna P. Singh; Shailendra K. Gupta; Rajeev Jain; Ratnasekhar Ch; R. C. Murthy (pp. 341-349).

The present communication describes the preparation and evaluation of a molecularly imprinted polymer (MIP) as a solid-phase extraction (SPE) sorbent and simultaneous ethyl chloroformate (ECF) derivatization and pre-concentration by dispersive liquid–liquid microextraction (DLLME) for the analysis of t,t-muconic acid (t,t-MA) in urine samples using gas chromatography–mass spectrometry. The imprinting polymer was prepared using methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linker, 2,2-azobisisobutyronitrile as the initiator and t,t-MA as a template molecule. The imprinted polymer was evaluated for its use as a SPE sorbent by comparing both imprinted and non-imprinted polymers in terms of the recovery of t,t-MA from urine samples. Molecular modelling studies were performed in order to estimate the binding energy and efficiency of the MIP complex formed between the monomer and the t,t-MA. Various factors that can affect the extraction efficiency of MIP, such as the loading, washing and eluting conditions, were optimized; other factors that can affect the derivatization and DLLME pre-concentration were also optimized. MIP in combination with ECF derivatization and DLLME pre-concentration for t,t-MA exhibits good linearity, ranging from 0.125 to 2 μg mL⁻¹ (R ² = 0.9971), with limit of detection of 0.037 μg mL⁻¹ and limit of quantification of 0.109 μg mL⁻¹. Intra- and inter-day precision was found to be <6 %. The proposed method has been proven to be effective and sensitive for the selective pre-concentration and determination of t,t-MA in urine samples of cigarette smokers. Figure Graphical abstract for t,t-muconic acid analysis by using MISPE-DLLME followed by GC-MS analysis

Keywords: Muconic acid; Molecularly imprinted polymers; Gas chromatography–mass spectrometry; Molecular modelling; Ethyl chloroformate; Dispersive liquid–liquid microextraction

Identifying indoor environmental patterns from bioaerosol material using HPLC by Sarah J. R. Staton; Josemar A. Castillo; Thomas J. Taylor; Pierre Herckes; Mark A. Hayes (pp. 351-357).

A substantial portion of the atmospheric particle budget is of biological origin (human and animal dander, plant and insect debris, etc.). These bioaerosols can be considered information-rich packets of biochemical data specific to the organism of origin. In this study, bioaerosol samples from various indoor environments were analyzed to create identifiable patterns attributable to a source level of occupation. Air samples were collected from environments representative of human high-traffic- and low-traffic indoor spaces along with direct human skin sampling. In all settings, total suspended particulate matter was collected and the total aerosol protein concentration ranged from 0.03 to 1.2 μg/m³. High performance liquid chromatography was chosen as a standard analysis technique for the examination of aqueous aerosol extracts to distinguish signatures of occupation compared to environmental background. The results of this study suggest that bioaerosol “fingerprinting” is possible with the two test environments being distinguishable at a 97 % confidence interval. Figure Generalized schematic of human debris-based occupation detection. The various human occupation-related aerosolized material are collected via the filtration assembly along with other non-related aerosolized material. The collected material was then analyzed for total protein concentration as well as coarsely separated to generated unique pattern profiles. These profiles are information-rich enough to identify human occupation in an indoor space

Keywords: Bioaerosols; Environmental monitoring; Environmental pattern recognition; HPLC; Separation

Evaluation of gel electrophoresis techniques and laser ablation–inductively coupled plasma-mass spectrometry for screening analysis of Zn and Cu-binding proteins in plankton by Maria S. Jiménez; L. Rodriguez; Juan R. Bertolin; Maria T. Gomez; Juan R. Castillo (pp. 359-368).

The determination of metal-binding proteins in plankton is important because of their involvement in photosynthesis, which is fundamental to the biogeochemical cycle of the oceans and other ecosystems. We have elaborated a new strategy for screening of Cu and Zn-containing proteins in plankton on the basis of separation of proteins by use of Blue-Native PAGE (BN-PAGE), which entails use of a non-denaturing Tris–tricine system and detection of metals in the proteins by laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS). For comparison, denaturing PAGE based on Tris–glycine and Tris–tricine systems and Anodic-Native PAGE have also been investigated. A large number of protein bands with MW between 20 and 75 kDa were obtained by use of Tris–glycine PAGE but detection of metals by LA–ICP–MS was unsuccessful because of loss of metals from the proteins during the separation process. Different protein extraction, purification, and preconcentration methods were evaluated, focussing on both issues—achieving the best extraction and characterization of the proteins while maintaining the integrity of metal–protein binding in the plankton sample. Use of 25 mmol L⁻¹ Tris–HCl and a protease inhibitor as extraction buffer with subsequent ultrafiltration and acetone precipitation was the most efficient means of sample preparation. Two Cu and Zn proteins were detected, a protein band corresponding to a MW of 60 kDa and another poorly resolved band with a MW between 15 and 35 kDa.

Keywords: Gel electrophoresis; Laser ablation; ICP–MS; Metal-binding proteins; Plankton

The National Institute of Standards and Technology ambient level methane in air Standard Reference Material historical record by George C. Rhoderick (pp. 369-375).

The National Institute of Standards and Technology (NIST) has been certifying lots, or series, of Standard Reference Materials (SRMs) containing ambient level methane in air for over 40 years. The historical record contains six traditional series of SRM 1658 (1 μmol mol⁻¹), five of SRM 1660 (4 μmol mol⁻¹), and seven of SRM 1659 (10 μmol mol⁻¹) methane in air. All series of any one particular SRM can be linked to each other through the historical suites of gravimetric primary standard mixtures (PSMs) developed at NIST. One gas mixture cylinder from a series is chosen as the lot standard (LS), retained and held at NIST, and periodically compared to the PSMs to assure its stability. Recently, 6 of the original 18 LS still in service in the Gas Metrology Group inventory, and cylinder samples held at NIST from 6 other SRM lots, were analyzed against a newly prepared suite of PSMs using cavity ring-down spectroscopy. Data were analyzed using a generalized least squares linear regression. The results indicate that, within the original 95 % confidence intervals, the methane concentration has remained the same for all the SRM LS and lot samples. The current predicted concentrations of the LS and samples for SRMs 1659 and 1660 are within 0.002 to 0.051 μmol mol⁻¹, or ≤0.5 %, relative of the original certificate value. SRM 1658 LS and samples are within 0.0001 to 0.0023 μmol mol⁻¹, or ≤0.2 % relative. These results illustrate the consistency, repeatability, and stability of these methane in air SRMs over the historical 35+-year record. It also demonstrates that the historical gravimetric primary methane in air suites have remained accurate and consistent over time.

Keywords: Standard Reference Materials; Methane; Primary gravimetric standard

On-line solid-phase microextraction of triclosan, bisphenol A, chlorophenols, and selected pharmaceuticals in environmental water samples by high-performance liquid chromatography–ultraviolet detection by Dalho Kim; Jungho Han; Yongwook Choi (pp. 377-387).

A method using on-line solid-phase microextraction (SPME) on a carbowax-templated fiber followed by liquid chromatography (LC) with ultraviolet (UV) detection was developed for the determination of triclosan in environmental water samples. Along with triclosan, other selected phenolic compounds, bisphenol A, and acidic pharmaceuticals were studied. Previous SPME/LC or stir-bar sorptive extraction/LC-UV for polar analytes showed lack of sensitivity. In this study, the calculated octanol–water distribution coefficient (log D) values of the target analytes at different pH values were used to estimate polarity of the analytes. The lack of sensitivity observed in earlier studies is identified as a lack of desorption by strong polar–polar interactions between analyte and solid-phase. Calculated log D values were useful to understand or predict the interaction between analyte and solid phase. Under the optimized conditions, the method detection limit of selected analytes by using on-line SPME-LC-UV method ranged from 5 to 33 ng L⁻¹, except for very polar 3-chlorophenol and 2,4-dichlorophenol which was obscured in wastewater samples by an interfering substance. This level of detection represented a remarkable improvement over the conventional existing methods. The on-line SPME-LC-UV method, which did not require derivatization of analytes, was applied to the determination of TCS including phenolic compounds and acidic pharmaceuticals in tap water and river water and municipal wastewater samples. Figure Schematic diagram of the On-line solid-phase microextraction

Keywords: Triclosan; Chlorophenols; Acidic pharmaceuticals; Bisphenol A; Solid-phase microextraction; Octanol–water distribution coefficient

Gas chromatography–triple-quadrupole mass spectrometry for analysis of selected polyhalogenated pollutants in plants. Comparison of extraction methods by Rosa Ana Pérez; José Luis Tadeo; Beatriz Albero; Esther Miguel; Consuelo Sánchez-Brunete (pp. 389-400).

Different extraction methods, followed by gas chromatography coupled to triple quadrupole mass spectrometry, were evaluated for simultaneous extraction of seven polychlorinated biphenyls (PCBs) and six polybrominated diphenyl ethers (PBDEs) from common weeds. Pressurized liquid extraction (PLE) with in-cell clean-up, ultrasound-assisted extraction (UAE) with in-column clean-up, and UAE with dispersive solid-phase extraction (dSPE) clean-up were evaluated and compared. In-cell clean-up with 4 g Florisil and 0.5 g graphitized carbon black (GCB) and two extraction cycles of 10 min with n-hexane–ethyl acetate 80:20 (v/v) at 60 °C were used for the PLE procedure. UAE with in-column clean-up was conducted under conditions similar to those reported for the PLE method whereas in UAE with dSPE clean-up purification of the extract was performed after extraction using primary and secondary amine sorbent (PSA) and GCB. Recovery from 82 to 104 % was obtained for all the compounds by PLE whereas, in general, lower extraction efficiency was obtained by UAE with in-column clean-up (especially for BDE-17 and BDE-183, for which recovery was 70 and 41 %, respectively) and by UAE with dSPE clean-up, for which the main drawback is that BDE-183 cannot be extracted. Finally, PLE was used for analysis of PCBs and PBDEs in different plants (Lolium rigidum, Lactuca serriola, Malva sylvestris, and Verbascum thapsus) collected from residential and/or rural areas of Madrid (Spain). Several of the analyzed compounds were detected at low levels in these plants, but only PCB-153 could be quantified. Figure Analysis of PCBs and PBDEs from plants

Keywords: Polybrominated diphenyl ethers; Polychlorinated biphenyls; Plants; Gas chromatography–triple quadrupole mass spectrometry; Pressurized liquid extraction; Ultrasound-assisted extraction

Environmentally friendly analysis of emerging contaminants by pressurized hot water extraction–stir bar sorptive extraction–derivatization and gas chromatography–mass spectrometry by Marina G. Pintado-Herrera; Eduardo González-Mazo; Pablo A. Lara-Martín (pp. 401-411).

This work describes the development, optimization, and validation of a new method for the simultaneous determination of a wide range of pharmaceuticals (beta-blockers, lipid regulators…) and personal care products (fragrances, UV filters, phthalates…) in both aqueous and solid environmental matrices. Target compounds were extracted from sediments using pressurized hot water extraction followed by stir bar sorptive extraction. The first stage was performed at 1,500 psi during three static extraction cycles of 5 min each after optimizing the extraction temperature (50–150 °C) and addition of organic modifiers (% methanol) to water, the extraction solvent. Next, aqueous extracts and water samples were processed using polydimethylsiloxane bars. Several parameters were optimized for this technique, including extraction and desorption time, ionic strength, presence of organic modifiers, and pH. Finally, analytes were extracted from the bars by ultrasonic irradiation using a reduced amount of solvent (0.2 mL) prior to derivatization and gas chromatography–mass spectrometry analysis. The optimized protocol uses minimal amounts of organic solvents (<10 mL/sample) and time (≈8 h/sample) compared to previous existing methodologies. Low standard deviation (usually below 10 %) and limits of detection (sub-ppb) vouch for the applicability of the methodology for the analysis of target compounds at trace levels. Once developed, the method was applied to determine concentrations of these compounds in several types of sample (wastewater, seawater, pore water, and sediment) from Cadiz Bay (SW Spain). To our knowledge, these findings represent the first information available on the presence of some of the target compounds in the marine environment.

Keywords: Pharmaceuticals; Personal care products; Pressurized hot water extraction (PHWE); Stir bar sorptive extraction (SBSE); Gas chromatography–mass spectrometry (GC-MS)

Determination of colloidal gold nanoparticle surface areas, concentrations, and sizes through quantitative ligand adsorption by Manuel Gadogbe; Siyam M. Ansar; Guoliang He; Willard E. Collier; Jose Rodriguez; Dong Liu; I-Wei Chu; Dongmao Zhang (pp. 413-422).

Determination of the true surface areas, concentrations, and particle sizes of gold nanoparticles (AuNPs) is a challenging issue due to the nanoparticle morphological irregularity, surface roughness, and size distributions. A ligand adsorption-based technique for determining AuNP surface areas in solution is reported. Using a water-soluble, stable, and highly UV–vis active organothiol, 2-mercaptobenzimidazole (MBI), as the probe ligand, we demonstrated that the amount of ligand adsorbed is proportional to the AuNP surface area. The equivalent spherical AuNP sizes and concentrations were determined by combining the MBI adsorption measurement with Au³⁺ quantification of aqua regia-digested AuNPs. The experimental results from the MBI adsorption method for a series of commercial colloidal AuNPs with nominal diameters of 10, 30, 50, and 90 nm were compared with those determined using dynamic light scattering, transmission electron microscopy, and localized surface plasmonic resonance methods. The ligand adsorption-based technique is highly reproducible and simple to implement. It only requires a UV–vis spectrophotometer for characterization of in-house-prepared AuNPs.

Keywords: Gold nanoparticle (AuNP); Ligand adsorption; Surface area; Particle size

Sequence-specific electrochemical detection of double-strand PCR amplicons of PML/RARα fusion gene in acute promyelocytic leukemia by Yun Lei; Mei-juan Feng; Kun Wang; Li-qing Lin; Yuan-zhong Chen; Xin-hua Lin (pp. 423-428).

A novel electrochemical method for the sequence-specific detection of double-stranded polymerase chain reaction (PCR) products of PML/RARα fusion gene in acute promyelocytic leukemia (APL) was described in detail. Based on a “sandwich” sensing mode involving a pair of locked nucleic acids probes (capture probe and reporter probe), this DNA sensor exhibited excellent selectivity and specificity. The direct and quantitative analysis of double-stranded complementary was firstly performed by our sensor without the use of alkali, helicase enzymes, or denaturants. Finally, combining PCR technique with electrochemical detection scheme, PCR amplicons (191 bp) of the PML/RARα fusion gene were obtained and rapidly identified with a low detection limit of 79 fmol in the 100-μL hybridization system. The results clearly showed the power of sensor as a promising tool for the sensitive, specific, and portable detection of APL and other diseases.

Keywords: Electrochemical biosensor; PML/RARα fusion gene; Double-stranded DNA; Polymerase chain reaction

Glycated hemoglobin (HbA1c) measurement in frozen whole blood depends on baseline values of fresh samples by Luigi Liotta; Alessandra Di Franco; Mario Pazzagli; Michaela Luconi (pp. 429-434).

Glycated hemoglobin (HbA1c) has been recently adopted as a diagnostic marker of type 2 diabetes. However, its usage is currently limited to fresh blood samples. To allow retrospective HbA1c measurement in blood banks developed in large epidemic studies, here, we contribute to validate HbA1c assessment in frozen versus fresh blood samples from a cohort of diabetic/nondiabetic adult subjects. HbA1c was measured by HPLC in 237 fresh whole blood samples and on the same samples after a 12-month storage and a further 6-month-refrozen storage. Mean HbA1c ± SD in fresh, frozen, and refrozen samples was 6.9 ± 1.2, 6.6 ± 1.1, and 6.4 ± 1.0 % for the Diabetes Control and Complications Trial and 52 ± 13, 49 ± 12, and 46 ± 11 mmol/mol for the International Federation of Clinical Chemistry and Laboratory Medicine reference, respectively. A significant correlation was found between fresh/frozen and fresh/refrozen (R = 0.994 and 0.993, P < 0.001) samples. HbA1c relative error ratio (%RER) between frozen/refrozen and fresh samples significantly correlated with HbA1c and depended on fresh value range, increasing in the five HbA1c classes (<6.0, 6.0–6.5, 6.5–7, 7–8, ≥8 %, corresponding to <42, 42–48, 48–53, 53–64, ≥64 mmol/mol, P < 0.001). In particular, the 6.5 % (48 mmol/mol) HbA1c diagnostic cutoff of fresh samples identified two classes reflecting significant differences in %RER (2.8 ± 2.0 and 3.3 ± 1.7; P < 0.05) between frozen and fresh samples. In conclusion, our results demonstrate a high correlation between data from fresh and frozen samples, with a very limited %RER between the two measurements, which increases with baseline HbA1c levels. Accordingly, when analyzing biobank frozen specimens for diagnostic purpose, the effect of the HbA1c range should be taken into account. Figure HbA1c separation profile obtained by HPLC from a whole blood sample

Keywords: Glycated hemoglobin; Diabetes; Biobank; Whole blood; Frozen

Letter to the Editor regarding “Simultaneous determination of ∆⁹-tetrahydrocannabinol and 11-nor-9-carboxy-∆⁹-tetrahydrocannabinol in oral fluid using isotope dilution liquid chromatography tandem mass spectrometry” by Marie Fabritius; Christian Giroud (pp. 435-437).

Letter to the Editor regarding “Quantification of Paraquat, MPTP, and MPP+ in brain tissue using microwave-assisted solvent extraction (MASE) and high-performance liquid chromatography–mass spectrometry” by Brian Buckley (pp. 439-439).

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Analytical and Bioanalytical Chemistry (v.405, #1)