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Analytical and Bioanalytical Chemistry (v.399, #10)

Analytical and bioanalytical science in China by Lihua Zhang; Qiankun Zhuang; Yukui Zhang (pp. 3305-3306).

is a professor at Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Her research and interests focus on the development of new separation and identification methods for both qualitative and quantitative analysis of proteomes, by developing novel sample preparation techniques, multidimensional liquid-phase-based platforms, and coupling with mass spectrometry. is Director of the Analytical Chemistry Division, National Natural Science Foundation of China. He is an expert in electroanalytical chemistry, and is presently strongly involved in promoting the advancement of analytical and bioanalytical science in China is a member of the Chinese Academy of Sciences and a professor at Dalian Institute of Chemical Physics. His research is focused on the study of the fundamentals of chromatography, column chromatography, and instrumentation for chromatography, as well as applications in the bioanalysis of DNA, bile acids, sugars, and proteins. He is also member of the International Advisory Board of Analytical and Bioanalytical Chemistry.

Development of silica-based stationary phases for high-performance liquid chromatography by Hongdeng Qiu; Xiaojing Liang; Min Sun; Shengxiang Jiang (pp. 3307-3322).

Stationary phases are the basis of the development and application of high-performance liquid chromatography (HPLC). In this review we focused on the development of silica-based stationary phases, including the synthesis of silica gel and the application of silica in hydrophilic interaction chromatography (HILIC), reversed-phase liquid chromatography (RPLC), chiral separation chromatography, and ion chromatography. New stationary phases, advances in ionic liquid-modified silica, silica-based core-shell materials, and silica-based monolithic columns for HPLC are introduced separately.

Keywords: High-performance liquid chromatography; Silica stationary phase; Ionic liquid-modified silica; Organic–inorganic modification; Chiral separation

Capillary electrophoresis with electrochemiluminescence detection: fundamental theory, apparatus, and applications by Longhua Guo; FengFu Fu; Guonan Chen (pp. 3323-3343).

This review presents a comprehensive survey of recent progress on electrochemiluminescence (ECL) detection coupled with capillary electrophoresis (CE). The fundamental theories involved in CE-ECL, e.g., the mechanism involving both coreactant-based and inhibitor-based ECL, as well as the possible analytes to be detected by CE-ECL are summarized. Different schemes for the construction of CE-ECL apparatus, including methods for preparing the working electrode, approaches for addition of ECL reagents, ways to fabricate electrical decouplers, and factors affecting ECL efficiency are reviewed. Discussion of the literature related to the application of CE-ECL from January 2005 to September 2010 is sorted by the corresponding analyte matrixes, namely, the standard solution, urine, serum and plasma, and other matrixes. Finally, possible trends for CE-ECL in the near future are discussed. Online abstract figure CE-ECL for the detection of Ru(bpy)₃ ²⁺ labeled analytes Recent development showed that the coupling of electrochemiluminescence (ECL) to capillary electrophoresis (CE) can be used for the detection of a large number of analytes such as ECL coreactants, ECL inhibitors and Ru(bpy)₃ ²⁺ labeled analytes. This figure shows a typical example of using CE-ECL for the detection of protein labeled with Ru(bpy)₃ ²⁺.

Keywords: Capillary electrophoresis; Electrochemiluminescence; Fundamental theory; Apparatus; Application; Solid-state electrochemiluminescence

Porous monoliths: sorbents for miniaturized extraction in biological analysis by Li Xu; Zhi-Guo Shi; Yu-Qi Feng (pp. 3345-3357).

In this review the focus is on application of porous monoliths to miniaturized extraction of biological analysis, with emphasis on porous monolithic materials and different miniaturized extraction formats. The general approaches used to synthesize organic polymer and silica monolithic materials are highlighted, and their properties and applicability are described and compared. Several extraction formats, including in-tube microextraction, chip-based microextraction, tip-based microextraction, among others, are reviewed in depth. Figure A visual model of the miniaturized extraction based on porous monoliths

Keywords: Monolith; Miniaturization; Microextraction; Biological analysis

Reversed-phase depletion coupled with hydrophilic affinity enrichment for the selective isolation of N-linked glycopeptides by using Click OEG-CD matrix by Yanyan Zhao; Long Yu; Zhimou Guo; Xiuling Li; Xinmiao Liang (pp. 3359-3365).

Selective enrichment of glycopeptides is of great importance for protein glycosylation analysis using mass spectrometry since the signals of glycopeptides could be severely suppressed by the coexisting non-glycosylated peptides in the protein digest. In the present work, a strategy for N-linked glycopeptide enrichment through reversed-phase depletion coupled with hydrophilic affinity enrichment by applying the customized matrix named Click OEG-CD is developed. Compared with single hydrophilic interaction liquid chromatography (HILIC) mode, the strategy exhibited remarkably higher selectivity for N-linked glycopeptides. As many as 22, 18, and eight glycopeptides were detected in the glycopeptide fraction enriched with the strategy from the digests of human immunoglobulin G, horseradish peroxidase and bovine ribonuclease B, respectively. In addition, the strategy also showed high glycosylation microheterogeneity coverage for the enrichment of human α₁-acid glycoprotein glycopeptides. More than 170 glycopeptides covering all the glycosylation sites were detected in the enriched fraction. The revered-phase liquid chromatography depletion coupled with HILIC enrichment strategy by using Click OEG-CD matrix is expected to show more potential in further applications in glycosylation analysis. Figure A reversed-phase depletion coupled with HILIC glycopeptide enrichment strategy by using Click OEG-CD matrix was developed in the work

Keywords: Click OEG-CD; Glycopeptide enrichment; Glycoproteomics; Hydrophilic interaction liquid chromatography

Development of sample preparation method for auxin analysis in plants by vacuum microwave-assisted extraction combined with molecularly imprinted clean-up procedure by Yuling Hu; Yuanwen Li; Yi Zhang; Gongke Li; Yueqin Chen (pp. 3367-3374).

A novel sample preparation method for auxin analysis in plant samples was developed by vacuum microwave-assisted extraction (VMAE) followed by molecularly imprinted clean-up procedure. The method was based on two steps. In the first one, conventional solvent extraction was replaced by VMAE for extraction of auxins from plant tissues. This step provided efficient extraction of 3-indole acetic acid (IAA) from plant with dramatically decreased extraction time, furthermore prevented auxins from degradation by creating a reduced oxygen environment under vacuum condition. In the second step, the raw extract of VMAE was further subjected to a clean-up procedure by magnetic molecularly imprinted polymer (MIP) beads. Owing to the high molecular recognition ability of the magnetic MIP beads for IAA and 3-indole-butyric acid (IBA), the two target auxins in plants can be selectively enriched and the interfering substance can be eliminated by dealing with a magnetic separation procedure. Both the VMAE and the molecularly imprinted clean-up conditions were investigated. The proposed sample preparation method was coupled with high-performance liquid chromatogram and fluorescence detection for determination of IAA and IBA in peas and rice. The detection limits obtained for IAA and IBA were 0.47 and 1.6 ng/mL and the relative standard deviation were 2.3% and 2.1%, respectively. The IAA contents in pea seeds, pea embryo, pea roots and rice seeds were determined. The recoveries were ranged from 70.0% to 85.6%. The proposed method was also applied to investigate the developmental profiles of IAA concentration in pea seeds and rice seeds during seed germination. Figure Analytical procedure of auxins in plants

Keywords: Auxin; 3-Indole acetic acid; Vacuum microwave-assisted extraction; Magnetic molecularly imprinted polymer beads; Clean-up

Molecularly imprinted beads with double thermosensitive gates for selective recognition of proteins by Lei Qin; Xi-Wen He; Xia Yuan; Wen-You Li; Yu-Kui Zhang (pp. 3375-3385).

A new approach is reported on the use of poly(N-isopropylacrylamide) (PNIPAM)-coated molecularly imprinted beads (coated MIP beads) for controlling the release of protein. The coated MIP beads were composed of double layers, an internal thermosensitive lysozyme-imprinted layer, and an external PNIPAM layer. The coated MIP beads were prepared by two-step surface-initiated living-radical polymerization (SIP). In this systemic study, the coated MIP beads had good selectivity to the template protein (lysozyme) and temperature stimulus-responsive behavior, both of which were superior to those of MIP beads having a layer of thermosensitive lysozyme-imprinted polymer only. Using the coated MIP beads, reference proteins and the template lysozyme could be released separately at 38 °C and at 23 °C. The corresponding coated non-imprinted beads (coated NIP beads) did not have such double thermosensitive “gates” with specific selectivity for a particular protein. The proposed smart controlled imprinted system for protein is attractive for chemical carriers, drug-delivery system, and sensors. Figure Schematic illustration of the coated MIP beads with thermosensitive swelling/collapse phase transitions for selective adsorption of proteins

Keywords: Thermosensitive; Molecularly imprinted bead; Protein

Ionic liquid 1-butyl-3-methyl imidazolium tetrafluoroborate for shotgun membrane proteomics by Liangliang Sun; Dingyin Tao; Bin Han; Junfeng Ma; Guijie Zhu; Zhen Liang; Yichu Shan; Lihua Zhang; Yukui Zhang (pp. 3387-3397).

The solubility and digestion efficiency are two crucial factors that might affect the identification of integral membrane proteins (IMPs). In this work, 1% (v/v) ionic liquid (IL), 1-butyl-3-methyl imidazolium tetrafluoroborate (BMIM BF₄), added in NH₄HCO₃ buffer (pH 8.3), was applied as a sample preparation buffer for IMPs analysis. Compared to the commonly used sodium dodecyl sulfate and methanol methods, the number of identified IMPs from rat brain by microcolumn reversed phase liquid chromatography (μRPLC)-electrospray ionization tandem mass spectrometry (ESI-MS/MS) was improved by over three times, which might be due to the fact that BMIM BF₄ offered high solubilizing ability for IMPs and good compatibility for tryptic digestion. Furthermore, compared to Rapigest and urea methods, with BMIM BF₄ method, the number of identified IMPs from rat brain could be improved 25% and 80%, respectively, which might be contributed to the good solubilizing ability and high thermal stability of such IL. With the sample treated by BMIM BF₄ method, by 2D-nanoSCX-RPLC-ESI-MS/MS, 1,450 non-redundant proteins and 7,978 unique peptides were identified from rat brain, and 418 proteins contained at least one predicted transmembrane domain, with false discovery rates of less than 1% for peptide identification, and at least two identified unique peptides per protein. All these results demonstrate that the BMIM BF₄ method is of high potential for the large-scale identification of IMPs.

Keywords: Ionic liquid; 1-Butyl-3-methyl imidazolium tetrafluoroborate; Integral membrane proteins; Shotgun

Zirconium oxide aerogel for effective enrichment of phosphopeptides with high binding capacity by Liyuan Zhang; Jin Xu; Liangliang Sun; Junfeng Ma; Kaiguang Yang; Zhen Liang; Lihua Zhang; Yukui Zhang (pp. 3399-3405).

In this study, zirconium oxide (ZrO₂) aerogel was synthesized via a green sol–gel approach, with zirconium oxychloride, instead of the commonly used alkoxide with high toxicity, as the precursor. With such material, phosphopeptides from the digests of 4 pmol of β-casein with the coexistence of 100 times (mol ratio) BSA could be selectively captured, and identified by MALDI-TOF MS. Due to the large surface area (416.0 m² g⁻¹) and the mesoporous structure (the average pore size of 10.2 nm) of ZrO₂ aerogel, a 20-fold higher loading capacity for phosphopeptide, YKVPQLEIVPN[pS]AEER (MW 1952.12), was obtained compared to that of commercial ZrO₂ microspheres (341.5 vs. 17.87 mg g⁻¹). The metal oxide aerogel was further applied in the enrichment of phosphopeptides from 100 ng nonfat milk, and 17 phosphopeptides were positively identified, with a 1.5-fold improvement in phosphopeptide detection compared with previously reported results. These results demonstrate that ZrO₂ aerogel can be a powerful enrichment material for phosphoproteome study. Figure Great improvement on binding capacity of phosphopeptides by ZrO₂ aerogel

Keywords: ZrO₂ aerogel; Phosphopeptide; Enrichment; Mass spectrometry

In-column “click” preparation of hydrophobic organic monolithic stationary phases for protein separation by Xiangli Sun; Xiwen He; Langxing Chen; Yukui Zhang (pp. 3407-3413).

Two types of macroporous organic polymer monoliths based on glycidyl methacrylate (GMA), 4-vinylbenzyl chloride (VBC) and divinylbenzene (DVB) were prepared inside stainless-steel tubes. Azide functionalities were firstly introduced on the surfaces of poly(GMA-co-DVB) and poly(VBC-co-DVB) monoliths to provide reactive sites for click chemistry. With the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition, an in-column click-modification approach for covalent attachment of long alkyl chains onto polymer monoliths was developed. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by the scanning electron microscopy, mercury intrusion porosimeter, Fourier transform infrared spectroscopy, and elemental analyses, respectively. The chromatographic performances of the “clicked” stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min. Figure With the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition (CuAAC), an in-column click-modification approach for covalent attachment of long alkyl chains onto polymer monoliths based on poly(GMA-co-DVB)/poly(VBC-co-DVB) was developed. The “clicked” stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min

Keywords: Click chemistry; High-performance liquid chromatography; Polymer monolithic column; Protein separation

Protein separation on a polar-copolymerized C₈ stationary phase by Jun Dong; Long Yu; Xiuli Zhang; Xingya Xue; Zhimou Guo; Xinmiao Liang (pp. 3415-3421).

A novel polar-copolymerized C₈ stationary phase composed of octyl and chloropropyl (C₈-C₃Cl)-bonded silica has been developed for protein separation. Separation of standard proteins on this homemade column was investigated and compared with separation on a Waters Symmetry 300 C₄ column. Results indicated that the chromatographic performance of the homemade column in the separation of proteins was excellent. The new polar copolymerized C₈ stationary phase enabled glycoprotein separation with good resolution and reproducibility. It was also used for purification of ovalbumin glycoprotein in order to improve identification of the protein.

Keywords: Reversed-phase liquid chromatography; Polar-copolymerized; Protein separation; Glycoprotein

At-line coupling of magnetic-nanoparticle-based extraction with gel isoelectric focusing for protein analysis by Peng Dou; Zhen Liu (pp. 3423-3429).

Sample preparation is a crucial step for protein analysis. Functionalized magnetic nanoparticle (MNP)-based extraction has been developed to be a useful sample preparation technique for proteomic analysis. In this paper, we present a strategy for at-line coupling of MNP-based extraction (MNE) with gel isoelectric focusing (IEF). The key to the at-line combination is to use an anolyte or a catholyte as the desorbing agent. Thus, functionalized MNPs can be facilely at-line coupled with gel IEF, provided that the extraction/desorption process is pH-controlled. MNPs extracted with target proteins are added to the sample well, which can function as a natural adapter. Once a focusing electric field has been applied across the gel, proton ions migrating from the anolyte or hydroxide ions migrating from the catholyte can act as a desorbing agent, releasing the proteins from the MNE probes. The released proteins are consequently focused into distinct bands where the local pH equals their pI values. The at-line combination was well demonstrated with three types of functionalized nanoparticles: (1) phenylboronic acid functionalized MNPs for extracting glycoproteins through boronate affinity; (2) carboxyl-functionalized MNPs for extracting positively charged proteins through a weak cation exchange mechanism; and (3) amino-functionalized MNPs for extracting negatively charged proteins through a weak anion exchange mechanism. The at-line combination exhibited several significant advantages, including selectivity, sensitivity, and speed.

Keywords: At-line coupling; Extraction; Isoelectric focusing; Magnetic nanoparticles; Protein analysis

Proteomics investigation on aristolochic acid nephropathy: a case study on rat kidney tissues by Han-Zhi Wu; Lin Guo; Yuen-Fun Mak; Ning Liu; Wing-Tat Poon; Yan-Wo Chan; Zongwei Cai (pp. 3431-3439).

Prolonged intake of aristolochic acid (AA) has been shown to be associated with the development of certain renal disorders. Renal tubular atrophy and interstitial fibrosis are the early symptoms of AA nephropathy. The symptoms were observed in rats that were dosed with AA at a dosage of 10 mg/kg/day for 1 month. Apart from the renal tubular atrophy and interstitial fibrosis, AA–DNA adducts were detected in the rat kidney tissue. Differentiated proteins were identified in the kidney tissues from proteomics investigations. The upregulated proteins identified included ornithine aminotransferase, sorbitol dehydrogenase, actin, aspartoacylase, 3-hydroxyisobutyrate dehydrogenase, and peroxiredoxin-1. Downregulated proteins such as ATP synthase subunit β, glutamate dehydrogenase 1, regucalcin, glutamate–cysteine ligase regulatory subunit, dihydropteridine reductase, hydroxyacyl-coenzyme A dehydrogenase, voltage-dependent anion-selective channel protein 1, prohibitin, and adenylate kinase isoenzyme 4 were also identified. Several identified protein markers were found to have biological and medical significance. Figure Kidney fibrosis resulted from AA-exposure

Keywords: Aristolochic acid; Kidney disease; Protein biomarker; Proteomics

Stacking and determination of phenazine-1-carboxylic acid with low pK _a in soil via moving reaction boundaryformed by alkaline and double acidic buffers in capillary electrophoresis by Chong Sun; Xiao-Di Yang; Liu-Yin Fan; Wei Zhang; Yu-Quan Xu; Cheng-Xi Cao (pp. 3441-3450).

As shown herein, a normal moving reaction boundary (MRB) formed by an alkaline buffer and a single acidic buffer had poor stacking to the new important plant growth promoter of phenazine-1-carboxylic acid (PCA) in soil due to the leak induced by its low pK _a. To stack the PCA with low pK _a efficiently, a novel stacking system of MRB was developed, which was formed by an alkaline buffer and double acidic buffers (viz., acidic sample and blank buffers). With the novel system, the PCA leaking into the blank buffer from the sample buffer could be well stacked by the prolonged MRB formed between the alkaline buffer and blank buffer. The relevant mechanism of stacking was discussed briefly. The stacking system, coupled with sample pretreatment, could achieve a 214-fold increase of PCA sensitivity under the optimal conditions (15 mM (pH 11.5) Gly-NaOH as the alkaline buffer, 15 mM (pH 3.0) Gly-HCl-acetonitrile (20%, v/v) as the acidic sample buffer, 15 mM (pH 3.0) Gly-HCl as the blank buffer, 3 min 13 mbar injection of double acidic buffers, benzoic acid as the internal standard, 75 μm i.d. × 53 cm (44 cm effective length) capillary, 25 kV and 248 nm). The limit of detection of PCA in soil was decreased to 17 ng/g, the intra-day and inter-day precision values (expressed as relative standard deviations) were 3.17–4.24% and 4.17–4.87%, respectively, and the recoveries of PCA at three concentration levels changed from 52.20% to 102.61%. The developed method could be used for the detection of PCA in soil at trace level. Figure Schematic illustration of PCA stacking mechanism via a novel MRB system formed by alkaline buffer and double-acid buffers. (a) The initial arrangement in the stacking of PCA via the MRB formed with pH 11.5 Gly-NaOH buffer and double acidic buffers (one was the sample buffer in phase β and the another was blank buffer in phase β'); (b) the stacking of PCA in original sample plug and leak of PCA near the anodic end of sample plug into phase β'; (c) the stacking of PCA leaked into phase β' via the prolonged MRB formed between the alkaline buffer and the blank buffer. Phase α and α' contained pH 11.50 Gly-NaOH buffer, phase β had pH 3.00 Gly-HCl buffer with PCA, and phase β' held the pH 3.0 Gly-HCl blank buffer. The plus and minus symbols mean the anode and cathode, respectively. The arrows mean the movements of MRB, EOF, and negative-charged PCA

Keywords: Capillary electrophoresis; Moving reaction boundary; Phenazine-1-carboxylic acid; Stacking

DNA sensor by using electrochemiluminescence of acridinium ester initiated by tripropylamine by Yi He; Hao Zhang; Ying Chai; Hua Cui (pp. 3451-3458).

It was found that tripropylamine (TPA) could be used as a coreactant to initiate the electrochemiluminescence (ECL) of acridinium NHS ester (AE NHS) labels attached to DNA. The radicals generated in the electro-oxidation process of TPA reacted with AE NHS to form the excited N-methylacridone, giving rise to light emission. The AE/TPA ECL system was for the first time used as the detection system for developing an ECL-based DNA sensor. In the protocol, streptavidin-modified gold nanoparticles were firstly immobilized onto a thiol-treated gold electrode. The streptavidin could specifically interact with the biontinylated capture DNA. Afterwards, the target DNA and the AE-labeled report DNA were conjugated onto the electrode step by step due to the hybridization reactions, and a sandwich-type sensor was fabricated. The ECL signals of the sensor were obtained under pulse potential condition in alkaline solution containing 50.0 mmol L⁻¹ TPA. Under optimized experimental conditions, the linear range of the DNA sensor for the determination of the target DNA was from 5.0 × 10⁻¹⁵ to 5.0 × 10⁻¹² mol L⁻¹. The detection limit (S/N = 3) was 3.0 × 10⁻¹⁵ mol L⁻¹. Moreover, the sensor could specifically recognize the target DNA against one base-pair mismatched sequences, two base-pair mismatched sequences, and the noncomplementary sequences. It is of great application potential in clinic analysis.

Keywords: Electrochemiluminescence; DNA sensor; Acridinium ester; Gold nanoparticles

A methylation-stimulated DNA machine: an autonomous isothermal route to methyltransferase activity and inhibition analysis by Changfeng Zhu; Yanqin Wen; Hongzhen Peng; Yitao Long; Yao He; Qing Huang; Di Li; Chunhai Fan (pp. 3459-3464).

The operation of DNA nanomachines is generally triggered by either conformational changes of DNA nanostructure or external environmental stimuli. In the present study, we demonstrate an alternative driving force, DNA methylation, to stimulate DNA machine operation. DNA methylation changes neither DNA sequence and conformation nor external environment, however, blocks its cleavage by corresponding methylation-sensitive restriction endonuclease. We thus designed a strand displacement amplification DNA machine, which could be stimulated upon DNA methylation and then autonomously generates accumulated amounts of peroxidase-mimicking DNAzyme signaling machine products in an isothermal manner. The machine product DNAzyme could catalyze the H₂O₂-mediated oxidation of 2,2′-azino-bis(3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS²⁻) to a colored product ABTS^·−. This methylation-stimulated DNA machine was further used as a colorimetric assay for analysis of methyltransferases activities and screening of methylation inhibitors. As compared with classical methylation assay, this facile isothermal DNA machine avoids the introduction of methylation-specific polymerase chain reaction and radioactive labels, which might be employed as an effective tool for DNA methylation analysis.

Keywords: DNA machine; Methyltransferase activity; Inhibitor; Isothermal amplification

Comparison and characterization of polysaccharides from natural and cultured Cordyceps using saccharide mapping by Jia Guan; Jing Zhao; Kun Feng; De-Jun Hu; Shao-Ping Li (pp. 3465-3474).

Comparison and characterization of polysaccharides from natural and cultured Cordyceps on the basis of their chemical characteristics such as glycosidic linkages were performed for the first time using saccharide mapping. The results showed that polysaccharides from most of the natural and cultured Cordyceps had similar responses to enzymatic digestion. These polysaccharides mainly contained (1→4)-β-D-glucosidic linkages, and (1→4)-α-glucosidic, (1→6)-α-glucosidic, 1,4-β-D-mannosidic, as well as (1→4)-α-D-galactosiduronic linkages also existed in some polysaccharides. Especially, natural and cultured Cordyceps polysaccharides could be discriminated on the basis of high performance liquid chromatography profiles of pectinase hydrolysates, which is helpful to control the quality of polysaccharides from Cordyceps. Figure The procedure and typical profile of saccharide mapping.

Keywords: Cordyceps ; Polysaccharides; Enzymatic digestion; Saccharide mapping; High-performance size-exclusion chromatography

Identification of the neuronal effects of ethanol on C. elegans by in vivo fluorescence imaging on a microfluidic chip by Ying Wang; Jingjing Wang; Wei Du; Xiao Jun Feng; Bi-Feng Liu (pp. 3475-3481).

Caenorhabditis elegans (C. elegans) is a well-established model organism for investigating the correlations between behavioral and neuronal activities. Here, we demonstrated a microfluidic-based method that allowed stimulation-based neuronal analysis of immobilized C. elegans for identifying the neuronal effects of ethanol on the chemosensory responses of the right ASE (ASER) neuron. A one-piece microvalve was developed for the immobilization of C. elegans. Stimulations were realized by interface shifting of laminar flows. Well-fed transgenic worms expressing the calcium indicator G-CaMP in ASER neurons were used for in vivo fluorescence imaging. To evaluate the developed method, we first studied the effects of ethanol on the ASER neurons in response to a single NaCl stimulus. Results indicated that ethanol acutely suppressed the ON responses of ASER neurons to NaCl rather than the OFF response. Further studies of the adaptation of ASER neurons in response to NaCl and in the presence of ethanol suggested that ethanol interfered with the adaptation of neurons. The developed method exhibited the advantages of ease of operation and high throughput. We expect this new method to open up a new avenue for investigating the correlations between the behavioral and neuronal activities of C. elegans.

Keywords: Microfluidic chip; Ethanol; C. elegans ; ASER neuron; Fluorescence imaging

Characterisation of permanent markers by pyrolysis gas chromatography–mass spectrometry by Inez D. van der Werf; Giulia Germinario; Francesco Palmisano; Luigia Sabbatini (pp. 3483-3490).

Pyrolysis gas chromatography–mass spectrometry (PyGC-MS) was used as a rapid method for the characterization of permanent marker ink. Twenty-four samples of various colours purchased from different manufacturers were characterised. Four main typologies of polymer-binding medium could be distinguished on the basis of the pyrolysis products, and differentiation between permanent markers of different manufacturers could be accomplished. For some permanent marker samples, PyGC-MS analysis allowed pigment identification as well.

Keywords: Pyrolysis; GC-MS; Permanent marker; Ink

MALDI-MS and multivariate analysis for the detection and quantification of different milk species by Nicoletta Nicolaou; Yun Xu; Royston Goodacre (pp. 3491-3502).

The extensive consumption of milk and dairy products makes these foodstuffs targets for potential adulteration with financial gains for unscrupulous producers. Such practices must be detected as these can impact negatively on product quality, labelling and even health. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) is a potentially useful technique, with proven abilities in protein identification and more recently through the use of internal standards for quantification purposes of specific proteins or peptides. In the current work, we therefore aim to explore the accuracy and attributes of MALDI-ToF-MS with chemometrics for the detection and quantification of milk adulteration. Three binary mixtures containing cows' and goats', cows' and sheep's, and goats' and sheep's milk and a fourth tertiary mixture containing all types of milk were prepared and analysed directly using MALDI-ToF-MS. In these mixtures, the milk concentrations of each milk varied from 0% to 100% in 5% steps. Multivariate statistical methods including partial least squares (PLS) regression and non-linear Kernel PLS regression were employed for multivariate calibration and final interpretation of the results. The results for PLS and KPLS were encouraging with between 2% and 13% root mean squared error of prediction on independent data; KPLS slightly outperformed PLS. We believe that these results show that MALDI-ToF-MS has excellent potential for future use in the dairy industry as a rapid method of detection and enumeration in milk adulteration.

Keywords: Bioanalytical methods; Chemometrics/statistics; Foods/beverages; Mass spectrometry/MALDI-MS

Sample amount alternatives for data adjustment in comparative cyanobacterial metabolomics by Jan Huege; Leonard Krall; Marie-Caroline Steinhauser; Patrick Giavalisco; Rosmarie Rippka; Nicole Tandeau de Marsac; Dirk Steinhauser (pp. 3503-3517).

Here we describe an integrative protocol for metabolite extraction and the measurement of three cellular constituents, chlorophyll a, total protein, and glycogen from the same small volume of cyanobacterial cultures that can be used as alternative sample amount parameters for data adjustment in comparative metabolome studies. We conducted recovery experiments to assess the robustness and reproducibility of the measurements obtained for the cellular constituents. Also, we have chosen three profile-intrinsic parameters derived from gas chromatography-mass spectrometry (GC/MS) data in order to test their utility for spectral data adjustment. To demonstrate the relevance of these six parameters, we analyzed three cyanobacteria with greatly different morphologies, comprising a unicellular, a filamentous, and a filamentous biofilm-forming strain. Comparative analysis of GC/MS data from cultures grown under standardized conditions indicated that adjustment of the corresponding metabolite profiles by any of the measured cellular constituents or chosen intrinsic parameters led to similar results with respect to sample cohesion and strain separation. Twenty-one metabolites significantly enriched for the carbohydrate and amine superclasses are mainly responsible for strain separation, with a majority of the remaining metabolites contributing to sample group cohesion. Therefore, we conclude that any of the parameters tested in this study can be used for spectral data adjustment of cyanobacterial strains grown under controlled conditions. However, their use for the differentiation between different stresses or physiological states within a strain remains to be shown. Interestingly, both the adjustment approaches and statistical tests applied effected the detection of metabolic differences and their patterns among the analyzed strains. Figure Two-dimensional polar plot representation of the metabolite patterns among the strains analyzed. The associated ANOVA p-values are based on spectral data adjusted by the selected ion counts for all metabolites (sIC). Symbols positioned on the dashed black lines indicate that the mean pool size differences for a given strain lie halfway between those determined for the other two strains. Symbols positioned on the solid black lines represent metabolite mean pool size differences of two strains which are exactly the same. The distance to the center (radius) reflects the ANOVA p-value associated with a metabolite pattern. The solid line between dots in the small pattern plots is drawn to aid interpretation. The strains are abbreviated as follows: Syn - Synechocystis sp. PCC 6803, Nos - Nostoc sp. PCC 7120, and Osc - Oscillatoria sp. PCC 7112

Keywords: Synechocystis ; Nostoc ; Oscillatoria ; Comparative metabolomics; Spectral adjustment

Metabolite profiling of sucrose effect on the metabolism of Melissa officinalis by gas chromatography-mass spectrometry by Sooah Kim; Min Hye Shin; Md. Aktar Hossain; Eun Ju Yun; Hojoung Lee; Kyoung Heon Kim (pp. 3519-3528).

The effect of sugar on plant metabolism, which is known to be similar to hormone-like signaling, was metabolomically studied using Melissa officinalis (lemon balm). The metabolite profiles of M. officinalis treated with sucrose were analyzed by gas chromatography-mass spectrometry (GC-MS) and principal component analysis (PCA). A total of 64 metabolites from various chemical classes including alcohols, amines, amino acids, fatty acids, inorganic acids, organic acids, phosphates, and sugars were identified by GC-MS. Three groups treated with different sucrose concentrations were clearly separated by PCA of their metabolite profiles, indicating changes in the levels of many metabolites depending on the sucrose concentration. Metabolite profiling revealed that treatment with a higher sucrose level caused an increase in the levels of metabolites such as sugars, sugar alcohols, and sugar phosphates, which are related to the glycolytic pathway of M. officinalis. Furthermore, proline and succinic acid, which are associated with the proline-linked pentose phosphate pathway, the shikimic acid pathway, and the biosynthesis of phenylpropanoids, also increased with increasing sucrose concentration. Therefore, these metabolic changes induced by sucrose ultimately led to the increased production of flavonoids such as caffeic acid via the biosynthetic pathway of phenylpropanoids. This study demonstrated that the abundance changes in some primary and secondary metabolites were somewhat interlocked with each other in response to sucrose.

Keywords: Metabolite profiling; Melissa officinalis ; Sucrose; Gas chromatography-mass spectrometry

Glycation and oxidation of histones H2B and H1: in vitro study and characterization by mass spectrometry by Sofia Guedes; Rui Vitorino; Maria R. M. Domingues; Francisco Amado; Pedro Domingues (pp. 3529-3539).

Among the post-translational modifications, oxidation and glycation are of special interest, especially in diseases such as diabetes, and in aging. The synergistic interaction between glycation and oxidation, also known as “glycoxidation” is highly relevant due to its involvement in the production of deleterious changes at the molecular level. Non-enzymatic damage to nuclear proteins has potentially severe consequences for the maintenance of genomic integrity [54]. In this report, we study glycated histones and its in vitro oxidation. Data concerning the modifications that occurred in the histones were obtained by analysis of enzymatic digests (Glu-C and Arg-C) of unmodified and glycated histones, obtained before and after oxidation. Analysis was then performed using a MALDI-MS/MS-based approach combined with nano liquid chromatography. This approach allowed us to identify histone H2B and H1 specific-sites of oxidation and to distinguish the most affected residues for each histone. The results showed the occurrence of a cumulative effect of oxidative damage in the glycated histones when subjected to in vitro oxidation, suggesting that structural changes caused by glycation induces histones to a pro-oxidant state. Comparing the data of oxidized glycated histones with data from unmodified oxidized histones, using the same model of oxidation, the results clearly show that these oxidative modifications occur earlier and more extensively in glycated histones. Furthermore, the results pointed to an increased oxidative damage in the vicinity of the glycated residues.

Keywords: Histones; Glycation; Protein oxidation; Mass spectrometry

Assessment of salsolinol N-methyltransferase activity in rat peripheral lymphocytes by liquid chromatography–electrospray time-of-flight mass spectrometry by Yongqian Zhang; Lin Wang; Xiaoling Mu; Jinyan Duan; Yong Zhu; Hong Qing; Yujuan Li; Shengyuan Xiao; Yulin Deng (pp. 3541-3545).

Salsolinol N-methyltranseferase (SNMT), which may play a crucial role in the pathogenesis of Parkinson’s diseases (PD), is a key enzyme to metabolize salsolinol into N-methylsalsolinol that is a neurotoxin specific to dopaminergic neurons. A sensitive method for the quantitative determination of SNMT activity in rat peripheral lymphocytes was developed and validated using liquid chromatography–electrospray with time-of-flight mass spectrometry (LC-ESI-TOF). The calibration curve was linear over the range of 7.40–368.80 nM, with 7.40 nM of the lower limit of quantification. The inter-day and intra-day precisions and accuracy for all samples were acceptable. The validated method was successfully applied for the determination of SNMT activity in both the substantia nigra (SN) and peripheral lymphocytes of a unilateral 6-hydroxydopamine-lesion model of Parkinson’s disease in rats. The SNMT activity in the peripheral lymphocytes treated with the 6-hydroxydopamine was significantly increased compared with the control and sham-operated groups, which was coincident with the alteration of SNMT activity in the SN. Our results might indicate that SNMT activity may become a potential clinical marker for PD.

Keywords: Salsolinol N-methyltransferase; N-Methylsalsolinol; LC-ESI-TOF; Peripheral lymphocytes; 6-OHDA

Formation of arenicin-1 microdomains in bilayers and their specific lipid interaction revealed by Z-scan FCS by Radek Macháň; Martin Hof; Tatsiana Chernovets; Maxim N. Zhmak; Tatiana V. Ovchinnikova; Jan Sýkora (pp. 3547-3554).

Z-scan fluorescence correlation spectroscopy (FCS) is employed to characterize the interaction between arenicin-1 and supported lipid bilayers (SLBs) of different compositions. Lipid analogue C8-BODIPY 500/510C5-HPC and ATTO 465 labelled arenicin-1 are used to detect changes in lipid and peptide diffusion upon addition of unlabelled arenicin-1 to SLBs. Arenicin-1 decreases lipid mobility in negatively charged SLBs. According to diffusion law analysis, microdomains of significantly lower lipid mobility are formed. The analysis of peptide FCS data confirms the presence of microdomains for anionic SLBs. No indications of microdomain formation are detected in SLBs composed purely of zwitterionic lipids. Additionally, our FCS results imply that arenicin-1 exists in the form of oligomers and/or aggregates when interacting with membranes of both compositions.

Keywords: Arenicin-1; Supported lipid bilayers; Antibacterial peptide; Z-scan FCS; Lipid mobility

Electrospray ionization-ion mobility spectrometry as a detection system for three-phase hollow fiber microextraction technique and simultaneous determination of trimipramine and desipramine in urine and plasma samples by M. T. Jafari; M. Saraji; H. Sherafatmand (pp. 3555-3564).

A novel method based on three-phase hollow fiber microextraction technique (HF-LPME) coupled with electrospray ionization-ion mobility spectrometry (ESI-IMS) was developed for the simultaneous determination of two antidepressant drugs (trimipramine and desipramine) in urine and plasma samples. The effects of various parameters such as type of organic solvent, composition of donor and acceptor phase, stirring rate, salt addition, extraction time, and temperature were investigated. Under the optimized conditions, the relative standard deviation was in the range of 5–6%, and the method quantitation limit (MQL) of utilizing HF-LPME/ESI-IMS was 5 μg/L for both drugs. The relative recoveries obtained by the proposed method from urine and plasma samples were in the range 94% to 97% for trimipramine and 92% to 96% for desipramine. Finally, the feasibility of the proposed method was successfully confirmed by extraction and determination of trace amounts of trimipramine and desipramine in biological samples without any significant matrix effect. Figure Schematic presentation of the electrospray ionization-ion mobility spectrometry as a detection system for three-phase hollow fiber microextraction technique

Keywords: Antidepressant; Desipramine; Trimipramine; Three-phase microextraction; Ion mobility spectrometry

Determination of chlorinated solvents in industrial water and wastewater by DAI–GC–ECD by Marek Tobiszewski; Jacek Namieśnik (pp. 3565-3572).

A very simple and quick analytical method, based on direct aqueous injection, for determination of halogenated solvents in refinery water and wastewater, is described. There is a need to determine halogenated solvents in refinery water streams, because they may originate from several processes. There is also a need to develop methods enabling VOX to be determined in samples containing oil fractions. The method described enables simultaneous determination of 26 compounds with low detection limits (sub-μg L⁻¹) and excellent precision, especially for highly halogenated solvents. The matrix effects of four types of sample were evaluated—the method seemed to be relatively insensitive to variations in matrix composition. Deuterated 1,2-dichloroethane was used as internal standard and surrogate compound in quantitative analysis; application of isotopically labelled compounds is rarely reported when non-mass spectrometric detectors are used for analysis. Analysis of real samples showed that the most frequently detected compounds were dichloromethane and 1,2-dichloroethane.

Keywords: Gas chromatography; Trihalomethanes; Matrix effects; VOX; Isotopically labelled compounds

Nanoparticles in cigarette smoke; real-time undiluted measurements by a scanning mobility particle sizer by Wouter D. van Dijk; Simone Gopal; Paul T. J. Scheepers (pp. 3573-3578).

Cigarette smoke is a complex mixture of smoke constituents, often characterised by size-resolved particle distributions. Since descriptions of ultrafine particles <50 nm are absent, our aim was to explore the existence of these nanoparticles in fresh and undiluted cigarette smoke. We measured undiluted smoke particles real-time by a scanning mobility particle sizer with Faraday cup electrometer, integrated in our custom-made smoking machine. Cigarettes were smoked by 2 s puffs, 30 s puff intervals and 50 ml puff volume. We tested six different cigarettes (1–10 mg tar per cigarette) at ten particle size-ranges between 6 and 50 nm, and repeated measurements five times. The formation of nanoparticles in fresh cigarette smoke was observed over the entire range between 6 and 50 nm, and reproduced in all cigarettes. The highest mean yield was 8.8 × 10⁹ (SD = 1.1 × 10⁹) particles per cigarette at the largest particle size range by high-tar cigarettes. Nanoparticle counts appear to increase with particle size, claimed tar values and blocking of filter ventilation holes, and inversely with butt length. Fresh undiluted cigarette smoke contains large amounts of potentially toxic nanoparticles <50 nm. We recommend to further study nanoparticles in the characterisation of cigarette smoke.

Keywords: Nanoparticles; Cigarette smoke; SMPS + E; Particle size distribution

Transformations of polycyclic musks AHTN and HHCB upon disinfection with hypochlorite: two new chlorinated disinfection by-products (CDBP) of AHTN and a possible source for HHCB-lactone by Paul Kuhlich; Robert Göstl; Philip Teichert; Christian Piechotta; Irene Nehls (pp. 3579-3588).

In this work, the behavior of the polycyclic musks 6-acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-γ-2-benzopyran (HHCB) was investigated upon disinfection by using sodium hypochlorite as disinfectant in a model disinfection basin in order to find new disinfection by-products (DBP). In the case of AHTN, the carboxylic acid 3,5,5,6,8,8-hexamethyl-5,6,7,8-tetrahydronaphthalene-2-carboxylic acid (AHTN-COOH) was generated by a haloform reaction, being the origin for two new chlorinated DBPs. In the case of HHCB, disinfection via hypochlorite led to the HHCB-lactone. All reaction products and intermediates were synthesized and isolated. The relevant degradation mechanisms are discussed in detail.

Keywords: Musk; AHTN; HHCB; HHCB-lactone; Chlorination; Disinfection

Degradation product emission from historic and modern books by headspace SPME/GC–MS: evaluation of lipid oxidation and cellulose hydrolysis by Andrew J. Clark; Jesse L. Calvillo; Mark S. Roosa; David B. Green; Jane A. Ganske (pp. 3589-3600).

Volatile organic compounds emitted from a several decade series of bound periodicals (1859–1939) printed on ground wood paper, as well as historical books dating from the 1500s to early 1800s made from cotton/linen rag, were studied using an improved headspace SPME/GC–MS method. The headspace over the naturally aging books, stored upright in glass chambers, was monitored over a 24-h period, enabling the identification of a wide range of organic compounds emanating from the whole of the book. The detection of particular straight chain aldehydes, as well as characteristic alcohols, alkenes and ketones is correlated with oxidative degradation of the C₁₈ fatty acid constituency of paper. The relative importance of hydrolytic and oxidative chemistry involved in paper aging in books published between 1560 and 1939 was examined by comparing the relative abundances of furfural (FUR) a known cellulose hydrolysis product, and straight chain aldehydes (SCA) produced from the oxidation of fatty acids in paper. The relative abundance of furfural is shown to increase across the 379-year publication time span. A comparison of relative SCA peak areas across the series of books examined reveals that SCA emission is more important in the cotton/linen rag books than in the ground wood books.

Keywords: Paper; Cotton/linen rag; Fatty acid; Lipid; Aging; Oxidation

Classification and identification of organic binding media in artworks by means of Fourier transform infrared spectroscopy and principal component analysis by A. Sarmiento; M. Pérez-Alonso; M. Olivares; K. Castro; I. Martínez-Arkarazo; L. A. Fernández; J. M. Madariaga (pp. 3601-3611).

Fourier transform infrared spectroscopy is a powerful analytical technique to study organic materials. However, in Cultural Heritage, since the sample under analysis is always a complicated matrix of several materials, data analysis performed through peak-by-peak comparisons of sample spectra with those of standard compounds is a tedious method that does not always provide good results. To overcome this problem, a chemometric model based on principal component analysis was developed to classify and identify organic binding media in artworks. The model allows the differentiation of five families of binders: drying oils, waxes, proteins, gums, and resins, taking into account the absorption bands in two characteristic spectral windows: C–H stretching and carbonyl band. This new methodology was applied in the characterization of binders in three kinds of artworks: papers of historical, archeological, and artistic value, easel paintings, and polychromed stone-based sculptures. Figure Analysis of the binder in a wallpaper of the 19th century by means of FTIR spectroscopy and chemometrics

Keywords: Binder; FTIR; Artwork; PCA

Solid-state reference electrodes based on carbon nanotubes and polyacrylate membranes by F. Xavier Rius-Ruiz; Anna Kisiel; Agata Michalska; Krzystof Maksymiuk; Jordi Riu; F. Xavier Rius (pp. 3613-3622).

A novel potentiometric solid-state reference electrode containing single-walled carbon nanotubes as the transducer layer between a polyacrylate membrane and the conductor is reported here. Single-walled carbon nanotubes act as an efficient transducer of the constant potentiometric signal originating from the reference membrane containing the Ag/AgCl/Cl⁻ ions system, and they are needed to obtain a stable reference potentiometric signal. Furthermore, we have taken advantage of the light insensitivity of single-walled carbon nanotubes to improve the analytical performance characteristics of previously reported solid-state reference electrodes. Four different polyacrylate polymers have been selected in order to identify the most efficient reservoir for the Ag/AgCl system. Finally, two different arrangements have been assessed: (1) a solid-state reference electrode using photo-polymerised n-butyl acrylate polymer and (2) a thermo-polymerised methyl methacrylate:n-butyl acrylate (1:10) polymer. The sensitivity to various salts, pH and light, as well as time of response and stability, has been tested: the best results were obtained using single-walled carbon nanotubes and photo-polymerised n-butyl acrylate polymer. Water transport plays an important role in the potentiometric performance of acrylate membranes, so a new screening test method has been developed to qualitatively assess the difference in water percolation between the polyacrylic membranes studied. The results presented here open the way for the true miniaturisation of potentiometric systems using the excellent properties of single-walled carbon nanotubes.

Keywords: Solid-state reference electrodes; Carbon nanotubes; Polyacrylate membrane; Water transport

Highly sensitive and fast responsive fiber-optic modal interferometric pH sensor based on polyelectrolyte complex and polyelectrolyte self-assembled nanocoating by Mingjie Yin; Bobo Gu; Qiang Zhao; Jinwen Qian; Aping Zhang; Quanfu An; Sailing He (pp. 3623-3631).

A new fiber-optic pH sensor is demonstrated by coating negatively charged polyelectrolyte complex (PEC⁻) nanoparticles, made of sodium carboxymethyl cellulose and poly(diallyldimethylammonium chloride) (PDDA), and positively charged PDDA on the surface of a thin-core fiber modal interferometer (TCFMI) with a layer-by-layer (LbL) electrostatic self-assembly method. The fabricated TCFMI pH sensor has different transmission dip wavelengths under different pH values and shows high sensitivities of 0.6 nm/pH unit and −0.85 nm/pH unit for acidic and alkaline solutions, respectively, and short response time of 30–50 s. The LbL electrostatic self-assembly process of a PEC⁻/PDDA multilayer is traced by quartz crystal microbalance and shows a fast thickness growth. Atomic force microscopy shows the root mean square (RMS) surface roughness of electrostatic self-assembly nanocoating of polyelectrolyte complex/polyelectrolyte is much higher than that of polyelectrolyte/polyelectrolyte due to the larger size of PEC⁻ colloidal nanoparticles. The enhanced RMS surface roughness and thickness of the nanocoating can shorten the response time and raise the sensitivity of the TCFMI pH sensor, respectively. In addition, the TCFMI pH sensor has highly reversible performance and good durability.

Keywords: Electrostatic self-assembly; Optical fiber; pH sensor; Refractive index

Erratum to: Characterization and classification of the aroma of beer samples by means of an MS e-nose and chemometric tools by L. Vera; L. Aceña; J. Guasch; R. Boqué; M. Mestres; O. Busto (pp. 3633-3634).

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Analytical and Bioanalytical Chemistry (v.399, #10)

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Analytical and Bioanalytical Chemistry (v.399, #10)