Analytical and Bioanalytical Chemistry (v.399, #7)

Nuclear Overhauser effect challenge by Reinhard Meusinger (2303-2306).

Solution to Cleopatra's Cocktail challenge by Prudence J. Jones (2307-2307).

Analytical challenges in nanomedicine by Christine Kranz; Douglas C. Eaton; Boris Mizaikoff (2309-2311).
is currently heading the surface analytical group at the Institute of Analytical and Bioanalytical Chemistry at the University of Ulm, Ulm, Germany. In addition, she has been a member of the Center for Cell and Molecular Signaling at Emory University, School of Physiology since 2005. She is working in the field of scanning probe microscopy, in particular, scanning electrochemical microscopy (SECM), multifunctional scanning probes (e.g., combination AFM-SECM, IR-SECM, IR-AFM), and miniaturized amperometric biosensor technology, integrated microsystems, biomimetic sensors, and DNA sensing. Her research interests focus on the development of novel multifunctional scanning micro- and nanoprobe concepts, surface modification, and characterization with scanning probe techniques. In her function she is the scientific supervisor of the recently established FIB center UUlm. is a distinguished Professor and chair of Physiology at Emory University, Atlanta, GA, USA. The goal of his research is to examine the cellular signaling mechanisms, which regulate membrane ion transport and cellular homeostasis. To examine these signaling mechanisms, he applies contemporary methods of cellular and molecular biology including patch voltage clamp methods and expression of cloned signaling molecules in heterologous expression systems. There are three main areas of cellular signaling research in his laboratory: cellular responses involving steroid hormones and other lipid molecules, signaling mechanisms responsible for the responses of renal cells to growth factors and vasoactive substances like angiotensin II, and signaling mechanics that control fluid balance in the lungs. is currently Chaired Professor and Director of the Institute of Analytical and Bioanalytical Chemistry at the University of Ulm, Germany. He is also a member of the Center for Cell and Molecular Signaling at Emory University, Atlanta, GA. His research interests focus on optical sensors, biosensors, and biomimetic sensors operating in the mid-infrared spectral range, quantum cascade lasers, system miniaturization and integration based on micro- and nanofabrication, multifunctional scanning nanoprobes, scanning probe tip integrated nano(bio)sensors, focused ion beam techniques, development of chemical recognition layers for separation and sensing applications, chemometric data evaluation, environmental analytical chemistry, process analytical chemistry, and biomedical diagnostics.

The potential of microelectrode arrays and microelectronics for biomedical research and diagnostics by Ian L. Jones; Paolo Livi; Marta K. Lewandowska; Michele Fiscella; Branka Roscic; Andreas Hierlemann (2313-2329).
Planar microelectrode arrays (MEAs) are devices that can be used in biomedical and basic in vitro research to provide extracellular electrophysiological information about biological systems at high spatial and temporal resolution. Complementary metal oxide semiconductor (CMOS) is a technology with which MEAs can be produced on a microscale featuring high spatial resolution and excellent signal-to-noise characteristics. CMOS MEAs are specialized for the analysis of complete electrogenic cellular networks at the cellular or subcellular level in dissociated cultures, organotypic cultures, and acute tissue slices; they can also function as biosensors to detect biochemical events. Models of disease or the response of cellular networks to pharmacological compounds can be studied in vitro, allowing one to investigate pathologies, such as cardiac arrhythmias, memory impairment due to Alzheimer’s disease, or vision impairment caused by ganglion cell degeneration in the retina.
Keywords: Biochips; Biosensors; Electroanalytical methods; Microelectrode array; CMOS

Quantum dots as contrast agents for in vivo tumor imaging: progress and issues by Anthony J. Tavares; Lori Chong; Eleonora Petryayeva; W. Russ Algar; Ulrich J. Krull (2331-2342).
Quantum dots (QDs) have shown promise as imaging agents in cancer, heart disease, and gene therapy research. This review focuses on the design of QDs, and modification using peptides and proteins for mediated targeting of tissues for fluorescence imaging of tumors in vivo. Recent examples from the literature are used to illustrate the potential of QDs as effective imaging agents. The distribution and ultimate fate of QDs in vivo is considered, and considerations of designs that minimize potential toxicity are presented. Figure Angiogenic blood vessel
Keywords: Quantum dots; Fluorescence; Tumors; Imaging; In vivo; Toxicity

This paper reports an approach to detection of single nucleotide polymorphism based on special amplification assay and surface plasmon resonance biosensor technology. In this assay, a part of the target DNA is recognized by a probe (probe A) coupled with streptavidin–oligonucleotide (SON) complexes ex situ, and when the mixture is injected in the sensor, another part of the target DNA is recognized by a DNA probe (probe B) immobilized on the sensor surface. To achieve high sensitivity and specificity, the assay is optimized in terms of composition of SON complexes, probe design, and assay temperature. It is demonstrated that this approach provides high specificity (no response to targets containing single-mismatched bases) and sensitivity (improves sensor response to perfectly matched oligonucleotides by one order of magnitude compared to the direct detection method). The assay is applied to detection of a short synthetic analogue of TP53 containing a “hot spot”—single nucleotide mismatch frequently mutated in germ line cancer—at levels down to 40 pM.
Keywords: Surface plasmon resonance; TP53; Single nucleotide polymorphism; Point mutation; Amplification

Endothelial f-actin depolymerization enables leukocyte transmigration by Laura Isac; Gerold Thoelking; Albrecht Schwab; Hans Oberleithner; Christoph Riethmuller (2351-2358).
A demanding task of medicine is to understand and control the immune system. Central players in the cellular immune response are the leukocytes that leave the blood stream for host defense. Endothelial cells limit the emigration rate of leukocytes. Being located between blood and tissues, they permit or deny the passage. The exact mechanism of this process called diapedesis is not solved yet. Leukocytes can principally traverse either between cells (paracellularly) or directly through an individual endothelial cell (transcellularly). The transcellular way has recently gained experimental support, but it is not clear how the endothelial cytoskeleton manages to open and close a transmigratory channel. Atomic force microscopy was used to investigate the endothelial cytoskeleton. In order to directly access the leukocyte–endothelial interaction site, we applied a special protocol (“nanosurgery”). As a result, the endothelial cell turned out to become softer in a confined region strictly underneath the leukocyte. Fluorescence microscopy confirmed a depolymerization of the f-actin strands at the invasion site. Leukocytes dramatically rearrange the endothelial cytoskeleton to form transmigratory channels.
Keywords: Leukocyte; Endothelium; Transmigration; Cell mechanics; Atomic force microscopy; TIRF

Nanosensing of Fcγ receptors on macrophages by S. Furquan Ahmad; Lilia A. Chtcheglova; Barbara Mayer; Sergei A. Kuznetsov; Peter Hinterdorfer (2359-2367).
Determining the distribution of specific binding sites on biological samples with high spatial accuracy (in the order of several nanometer) is an important challenge in many fields of biological science. Combination of high-resolution atomic force microscope (AFM) topography imaging with single-molecule force spectroscopy provides a unique possibility for the detection of specific molecular recognition events. The identification and localization of specific receptor binding sites on complex heterogeneous biosurfaces such as cells and membranes are of particular interest in this context. Simultaneous topography and recognition imaging was used to unravel the nanolandscape of cells of the immune system such as macrophages. The most studied phagocytic receptors include the Fc receptors that bind to the Fc portion of immunoglobulins. Here, nanomapping of FcγRs (Fc receptors for immunoglobulin G (IgG)) was performed on fixed J774.A1 mouse macrophage cell surfaces with magnetically coated AFM tips functionalized with Fc fragments of mouse IgG via long and flexible poly(ethylene glycol) linkers. Because of possible AFM tip engulfment on living macrophages, appropriate cell fixation procedure leaving the binding activity of FcγRs practically intact was elaborated. The recognition maps revealed prominent spots (microdomains) more or less homogeneously distributed on the macrophage surface with the sizes from 4 to 300 nm. Typical recognition image contained about ∼4% of large clusters (>200 nm), which were surrounded by a massive number (∼50%) of small-size (4–30 nm) and the rest by middle-size (50, 150 nm) domains. These spots were detected from the decrease of oscillation amplitude during specific binding between Fc-coated tip and FcγRs on macrophage surfaces. In addition, the effect of osmotic swelling on the topographical landscape of macrophage surfaces and on the reorganization of FcγRs was investigated.
Keywords: FcγRs; Macrophages; Phagocytosis; TREC; AFM

Atomic force microscopy of microvillous cell surface dynamics at fixed and living alveolar type II cells by Elena Hecht; Shariq M. Usmani; Susanne Albrecht; Oliver H. Wittekindt; Paul Dietl; Boris Mizaikoff; Christine Kranz (2369-2378).
Scanning probe techniques enable direct imaging of morphology changes associated with cellular processes at life specimen. Here, glutaraldehyde-fixed and living alveolar type II (ATII) cells were investigated by atomic force microscopy (AFM), and the obtained topographical data were correlated with results obtained by scanning electron microscopy (SEM) and confocal microscopy (CM). We show that low-force contact mode AFM at glutaraldehyde-fixed cells provides complementary results to SEM and CM. Both AFM and SEM images reveal fine structures at the surface of fixed cells, which indicate microvilli protrusions. If ATII cells were treated with Ca2+ channel modulators known to induce massive endocytosis, changes of the cell surface topography became evident by the depletion of microvilli. Low force contact mode AFM imaging at fixed ATII cells revealed a significant reduction of the surface roughness for capsazepine and 2-aminoethoxydiphenyl-borate (CPZ/2-APB)-treated cells compared to untreated control cells (Rc of 99.7 ± 6.8 nm vs. Rc of 71.9 ± 4.6 nm for N = 22), which was confirmed via SEM studies. CM of microvilli marker protein Ezrin revealed a cytoplasmic localization of Ezrin in CPZ/2-APB-treated cells, whereas a submembranous Ezrin localization was observed in control cells. Furthermore, in situ AFM investigations at living ATII cells using low force contact mode imaging revealed an apparent decrease in cell height of 17% during stimulation experiments. We conclude that a dynamic reorganization of the microvillous cell surface occurs in ATII cells at conditions of stimulated endocytosis. Figure Low force contact mode AFM image of fixed AT(II) cells (false color image); inset: optical micrograph of the scanning AFM tip.
Keywords: Atomic force microscopy; ATII cells; Microvilli; Scanning electron microscopy; Confocal microscopy; Capsazepine (CPZ); 2-Aminoethoxydiphenyl-borate (2-APB); Lung; Epithelium

Laser microperforated biodegradable microbial polyhydroxyalkanoate substrates for tissue repair strategies: an infrared microspectroscopy study by Gary Ellis; Pilar Cano; María Jadraque; Margarita Martín; Laura López; Teresa Núñez; Enrique de la Peña; Carlos Marco; Leoncio Garrido (2379-2388).
Flexible and biodegradable film substrates prepared by solvent casting from poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) solutions in chloroform were microperforated by ultraviolet laser ablation and subsequently characterized using infrared (IR) microspectroscopy and imaging techniques and scanning electron microscopy (SEM). Both transmission synchrotron IR microspectroscopy and attenuated total reflectance microspectroscopy measurements demonstrate variations in the polymer at the ablated pore rims, including evidence for changes in chemical structure and crystallinity. SEM results on microperforated PHBHV substrates after cell culture demonstrated that the physical and chemical changes observed in the biomaterial did not hinder cell migration through the pores. Figure Composition showing visible and IR images of a microperforated PHBHV film, with IR spectra showing crystallinity differences between bulk film and pore rim, schematic of cell growth and propagation strategy and SEM image showing evidence of cell growth on the underside of the biodegradable substrate.
Keywords: FTIR microspectroscopy; Laser microfabrication; Poly(3-hydroxybutyrate-co-3-hydroxyvalerate); Biodegradable

The mystery of the discolored flints. New molecules turn prehistoric lithic artifacts blue by Andrea Tapparo; Gilberto Artioli; Ivana Angelini; Gabriella Favaro (2389-2393).
Prehistoric artifacts turning blue in the store rooms of the Natural History Museum in Verona, Italy recently raised serious issues for heritage materials conservation. Our analytical investigation showed that the unusual discoloration process of the flint tools is caused by the surface presence of at least three previously unknown pigmenting molecules of the triphenylmetane dyes class: 6-(bis(2,2,4-trimethyl-1,2-dihydroquinolin-6-yl)methylene)-2,2,4-trimethyl-2,6-dihydroquinolinium and its hydrogenated derivatives 2,2,4-trimethyl-6-((2,2,4-trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)(2,2,4-trimethyl-1,2-dihydroquinolin-6-yl)methylene)-2,6-dihydroquinolinium and 6-(bis(2,2,4-trimethyl-1,2,3,4-tetrahydroquinolin-6-yl)methylene)-2,2,4-trimethyl-2,6-dihydroquinolinium. The peculiar formation of the molecules is possibly catalyzed within the silica pore surface starting from a well-known rubber stabilizer 2,2,4-trimethyl-1,2-dihydroquinoline released by the plastic pads flooring the storing cabinets. The investigated reaction and its surprising blue product represent a case study of the application of modern materials science to conservation and a serious warning towards the unpredictable challenges faced in the preservation of our cultural heritage.
Keywords: Prehistoric artifacts; Blue flints; Triphenylmethane dyes; Mass spectrometry

A new method, based on the formation of gold nanoparticles (AuNPs) and spectrophotometric analysis, is proposed to determine total phenolic acids in virgin argan oil samples. These compounds have reducibility due to the presence of the phenol group in their molecular structure, and a redox reaction occurs in the presence of HAuCl4. The formation of AuNPs as a result of the redox reaction leading to colour changes can be visually observed, resulting in strong light signals that show absorption at 555 nm. As ferulic acid represents more than 95% of the total phenolic acid content of virgin argan oil, this compound was used as an adulteration marker to carry out the screening of samples for the evaluation of the authenticity of virgin argan oils. The analytical features of this screening method also allowed a low precision quantization of the quality of the product. Then, a reference HPLC-DAD/FD method was used to confirm the potential adulterated samples, as well as to provide a detailed quantitative analysis of the most representative phenolic compounds in the samples. The overall screening-confirmation strategy was validated by analysing pure virgin argan oil samples and argan oil samples adulterated with other commercial vegetable oils, demonstrating the reliability of the results. This approach is characterised by its simplicity, low cost, rapid information and responded to practical laboratories needs. Figure Analytical strategy for detecting virgin argan oil adulterations based on the use of AuNPs-absorbance measurements for the screening test (classification of samples) and HPLC as the confirmation method (for positive and non conclusive samples)
Keywords: Gold nanoparticles; Liquid chromatography; Phenolic compounds; Virgin argan oil; Adulteration

Functionalization of platinum nanoparticles for electrochemical detection of nitrite by Peng Miao; Min Shen; Limin Ning; Guifang Chen; Yongmei Yin (2407-2411).
In this work, a novel electrochemical method for nitrite detection by using functionalized platinum nanoparticles (PtNPs) is proposed. Firstly, a gold electrode is immobilized with 4-(2-aminoethyl)benzenamine. Then, PtNPs are modified with 5-[1, 2]dithiolan-3-yl-pentanoic acid [2-(naphthalene-1-ylamino)-ethyl]amide (DPAN). Consequently, in the presence of nitrite ions, Griess reaction occurs between 4-(2-aminoethyl)benzenamine on the electrode and DPAN on PtNPs, thus PtNPs are localized onto the electrode surface. So, PtNPs-electrocatalyzed reduction of H2O2 can be achieved to correlate the electrochemical signal with the concentration of nitrite ions. The linear concentration range can be as wide as 10–1,000 μM, while the detection limit is as low as 5 μM. The proposed method has been also successfully applied to the detection of nitrite with the local lake water, and the result is well consistent with that obtained by UV-visible spectrophotometric method. So, this method has potential use for monitoring nitrite in drinking water supplies in the future.
Keywords: Nitrite; Platinum nanoparticles; Griess reaction; Chemically modified electrode; Electrochemistry

Development of amperometric magnetogenosensors coupled to asymmetric PCR for the specific detection of Streptococcus pneumoniae by Susana Campuzano; María Pedrero; José L. García; Ernesto García; Pedro García; José M. Pingarrón (2413-2420).
A disposable magnetogenosensor for the rapid, specific and sensitive detection of Streptococcus pneumoniae is reported. The developed procedure involves the use of streptavidin-modified magnetic beads, a specific biotinylated capture probe that hybridizes with a specific region of lytA, the gene encoding the pneumococcal major autolysin, and appropriate primers for asymmetric polymerase chain reaction (PCR) amplification. Capture probes and amplicons specific for S. pneumoniae were selected by a careful analysis of all lytA alleles available. The selected primers amplify a 235-bp fragment of pneumococcal lytA. A detection limit (LOD) of 5.1 nM was obtained for a 20-mer synthetic target DNA without any amplification protocol, while the LOD for the asymmetric PCR amplicon was 1.1 nM. A RSD value of 6.9% was obtained for measurements carried out with seven different genosensors for 1.1-nM aPCR product. The strict specificity of the designed primers was demonstrated by aPCR amplification of genomic DNA prepared from different bacteria, including some closely related streptococci. Direct asymmetric PCR (daPCR), using cells directly from broth cultures of S. pneumoniae, showed that daPCR products could be prepared with as few as 2 colony-forming units (CFU). Furthermore, this methodology did not show any cross-reaction with closely related streptococci such as Streptococcus mitis (or Streptococcus pseudopneumoniae) even when present in the culture at concentrations up to 105 times higher than that of S. pneumoniae. Preliminary data for rapid detection of pneumococcus directly in clinical samples has shown that it is possible to discriminate between non-inoculated blood and urine samples and samples inoculated with only 103 CFU mL−1 S. pneumoniae. Figure A lytA-based magnetogenosensor for pneumococcal identification: The lytA gene, encoding the main pneumococcal autolysin, is a suitable target for an accurate diagnosis of the pneumococcal disease. Asymmetric PCR amplification with precisely designed primers together with amperometric measurements allows a rapid and accurate differentiation between S. pneumoniae and closely related streptococci (see picture)
Keywords: Streptococcus pneumoniae ; Biosensors; Gene technology; Bacterial identification; lytA gene

The determination of protein assembly size and relative molecular mass is currently of great importance in biochemical analysis. In particular, the technique of nanoelectrospray (nES) with a gas-phase electrophoretic mobility molecular analyzer (GEMMA) has received increased attention for such measurements. However, in order for the GEMMA technique to gain broader acceptance in protein analysis, it must be further evaluated and compared with other established bioanalytical techniques. In the present study, nES-GEMMA was evaluated for the analysis of a set of protein and protein complexes involved in the Sec and the bacterial type III secretion pathway of enteropathogenic Escherichia coli bacteria. The same set of proteins, isolated and purified using standard biochemical protocols, were also analyzed using multi-angle laser light scattering (MALLS) and quasi-elastic light scattering (QELS), following size exclusion chromatography. This allowed for direct comparisons between the three techniques. It was found that nES-GEMMA, in comparison to the more established MALLS and QELS techniques, offers several complementary advantages. It requires considerably less amount of material, i.e., nanogram vs. milligram amounts, and time per sample analysis, i.e., few minutes vs. tens of minutes. Whereas the determined size and relative molecular mass are similar between the compared methods, the electrophoretic diameters determined using nES-GEMMA seem to be systematically smaller compared to the hydrodynamic diameter derived by QELS. Some of the GEMMA technique disadvantages include its narrow dynamic range, limited by the fact that at elevated protein concentrations there is increased potential for the occurrence of nES-induced oligomers. Thus, it is preferred to analyze dilute protein solutions because non-specific oligomers are less likely to occur whereas biospecific oligomers remain detected. To further understand the formation of nES-oligomers, the effect of buffer concentration on their formation was evaluated. Also, nES-GEMMA is not compatible with all the buffers commonly used with MALLS and QELS. Overall, however, the nES-GEMMA technique shows promise as a high-throughput proteomics/protein structure tool. Figure NanoES-GEMMA electropherogram of SecA protein dimer
Keywords: Bioanalytical methods; Mass spectrometry/ICP-MS; Aerosols/particulates

An enantioselective liquid chromatographic method using two-phase hollow fiber liquid-phase microextraction (HF-LPME-HPLC) was developed for the determination of isradipine (ISR) enantiomers and its main metabolite (pyridine derivative of isradipine, PDI) in microsomal fractions isolated from rat liver. The analytes were extracted from 1 mL of microsomal medium using a two-phase HF-LPME procedure with hexyl acetate as the acceptor phase, 30 min of extraction, and sample agitation at 1,500 rpm. For the first time, ISR enantiomers and PDI were resolved. For this separation, a Chiralpak® AD column with hexane/2-propanol/ethanol (94:04:02, v/v/v) as the mobile phase at a flow rate of 1.5 mL min−1 was used. The column was kept at 23 ± 2 °C. The drug and metabolite detection was performed at 325 nm and the internal standard oxybutynin was detected at 225 nm. The recoveries were 23% for PDI and 19% for each ISR enantiomer. The method presented quantification limits (LOQ) of 50 ng mL−1 and was linear over the concentration range of 50–5,000 and 50–2,500 ng mL−1 for PDI and each ISR enantiomer, respectively. The validated method was employed to an in vitro biotransformation study of ISR using rat liver microsomal fraction showing that (+)-(S)-ISR is preferentially biotransformed.
Keywords: HF-LPME; Isradipine; Chiral HPLC; In vitro biotransformation; Microsomes

Three new polymeric chiral stationary phases were synthesized based on (1S,2S)-1,2-bis(2,4,6-trimethylphenyl)ethylenediamine, (1S,2S)-1,2-bis(2-chlorophenyl)ethylenediamine, and (1S,2S)-1,2-di-1-naphthylethylenediamine via a simple free-radical-initiated polymerization in solution. These monomers are structurally related to (1S,2S)-1,2-diphenylethylenediamine which is the chiral monomer used for the commercial P-CAP-DP polymeric chiral stationary phase (CSP). The performance of these three new chiral stationary phases were evaluated in normal phase high-performance liquid chromatography (HPLC) and supercritical fluid chromatography and the results were compared with those of the P-CAP-DP column. All three new phases showed enantioselectivity for a large number of racemates with a variety of functional groups, including amines, amides, alcohols, amino acids, esters, imines, thiols, and sulfoxides. In normal phase, 68 compounds were separated with 28 baseline separations (Rs ≥ 1.5) and in SFC, 65 compounds were separated with 24 baseline separations. In total 72 out of 100 racemates were separated by these CSPs with 37 baseline separations. Complimentary separation capabilities were observed for many analytes. The new polymeric CSPs showed similar or better enantioselectivities compared with the commercial column in both HPLC and SFC. However, faster separations were achieved on the new stationary phases. Also, it was shown that these polymeric stationary phases have good sample loading capacities while maintaining enantioselectivity. Figure Faster separations on new polymeric chiral stationary phases in HPLC
Keywords: Enantiomeric separations; Enantioselectivity; High-performance liquid chromatography; Polymeric chiral stationary phases; Supercritical fluid chromatography

Structural characterization of protonated gas-phase ions of cysteine and dopamine by infrared multiple photon dissociation (IRMPD) spectroscopy using a free electron laser in combination with theory based on DFT calculations reveals the presence of two types of protonated dimer ions in the electrospray mass spectra of the metabolites. In addition to the proton-bound dimer of each species, the covalently bound dimer of cysteine (bound by a disulfide linkage) has been identified. The dimer ion of m/z 241 observed in the electrospray mass spectra of cysteine has been identified as protonated cystine by comparison of the experimental IRMPD spectrum to the IR absorption spectra predicted by theory and the IRMPD spectrum of a standard. Formation of the protonated covalently bound disulfide-linked dimer ions (i.e. protonated cystine) from electrospray of cysteine solution is consistent with the redox properties of cysteine. Both the IRMPD spectra and theory indicate that in protonated cystine the covalent disulfide bond is retained and the proton is involved in intramolecular hydrogen bonding between the amine groups of the two cysteine amino acid units. For cysteine, the protonated covalently bound dimer (m/z 241) dominated the mass spectrum relative to the proton-bound dimer (m/z 243), but this was not the case for dopamine, where the protonated monomer and the proton-bound dimer were both observed as major ions. An extended conformation of the ethylammonium side chain of gas-phase protonated dopamine monomer was verified from the correlation between the predicted IR absorption spectra and the experimental IRMPD spectrum. Dopamine has the same extended ethylamine side chain conformation in the proton-bound dopamine dimer identified in the mass spectra of electrosprayed dopamine. The structure of the proton-bound dimer of dopamine is confirmed by calculations and the presence of an IR band due to the shared proton. The presence of the shared proton in the protonated cystine ion can be inferred from the IRMPD spectrum. Figure
Keywords: Bioanalytical methods; Laser spectroscopy; Mass spectrometry; Dopamine; Cysteine; Dimers

A new strategy using hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography and post-acquisition data mining techniques was developed and applied to the detection and characterization of degradation products of danofloxacin. The degradation products formed under different forced conditions were separated using an ODS-C18 column with gradient elution. Accurate full-scan MS data were acquired in the first run and processed with the combination of extracted ion chromatograms and LC-UV chromatograms. These processes were able to find accurate molecular masses of possible degradation products. Then, the accurate MS/MS data acquired through data-dependent analysis mode in another run facilitated the structural elucidations of degradation products. As a result, a total of 11 degradation products of danofloxacin were detected and characterized using the developed method. Overall, this analytical strategy enables the acquisition of accurate-mass LC/MS data, search of a variety of degradation products through the post-acquisition processes, and effective structural characterization based on elemental compositions of degradation product molecules and their product ions. The ability to measure degradation products via tandem mass spectrometry coupled with accurate mass measurement, all in only two experimental runs, is one of the most attractive features of this methodology. The results demonstrate that use of the LC/MS-IT-TOF approach appears to be rapid, efficient and reliable in structural characterization of drug degradation products. Figure An integrated method for degradation product detection and characterization using hybrid ion trap/time-of-flight mass spectrometry coupled with high-performance liquid chromatography (LC/MS-IT-TOF) and post-acquisition data mining techniques
Keywords: Ion trap/time-of-flight mass spectrometry; Degradation product; Accurate mass measurements; Structural characterization; Danofloxacin

New CZE-DAD method for honeybee venom analysis and standardization of the product by Zenon J. Kokot; Jan Matysiak; Bartosz Urbaniak; Paweł Dereziński (2487-2494).
The aim of this study was to develop a new precise and accurate CZE-DAD method for honeybee venom analysis using cytochrome c as an internal standard. The 64.5 cm total length, 56 cm effective length, 75 μm ID, and 360 μm OD uncoated fused-silica capillary was used. The samples were injected into the capillary under a 50-mbar pressure for 7 s. There were 15 kV of electric field across the capillary applied. The current intensity was 26 μA. The separation was carried out at 25 °C. The analysis was run with the normal electrode polarity. The following steps and parameters were taken into account for the validation of the developed method: selectivity, precision, accuracy, linearity, limit of detection and limit of quantitation. All steps of the validation procedure proved that the developed analytical procedure was suitable for its intended purpose. Possibly this was the first study in which several honeybee venom components were separated and five of them were identified by capillary zone electrophoresis. In addition, the developed method was applied for quantitative analysis of 38 honeybee venom samples. The content (relative to the dry venom mass) of analyzed peptides in honeybee venom samples collected in 2002–2007 was as follows: apamine from 0.93% to 4.34% (mean, 2.85 ± 0.79%); mast cell degranulating peptide (MCDP) from 1.46% to 4.37% (mean, 2.82 ± 0.64%); phospholipase A2 from 7.41% to 20.25% (mean, 12.95 ± 3.09%); melittin from 25.40% to 60.27%, (mean, 45.91 ± 9.78%). The results were compared with the experimental data obtained for the same venom samples analyzed earlier by the HPLC method. It was stated that HPCE and HPLC data did not differ significantly and that the HPCE method was the alternative for the HPLC method. Moreover, using the results obtained principal component analysis (PCA) was applied to clarify the general distribution patterns or similarities of four major honeybee venom constituents collected from two different bee strains in various months and years. PCA has shown that the strain of bee appears to be the only criteria for bee venom sample classification. Strong correlations between apamine, MCDP, phospholipase A2, and melittin were confirmed. These correlations have to be taken into account in the honeybee venom standardization. The developed method due to its simplicity can be easily automated and incorporated into routine operations both in the bee venom identification, quality control, and standardization of the product.
Keywords: Bioanalytical methods; Biological samples; Capillary electrophoresis; Chemometrics/statistics; HPLC; Natural products

Five ionic liquid-modified porous polymers with different imidazolium-based functional groups were obtained. A molecular imprinting technique was introduced to form the ordered functional groups in the porous structure. The adsorption isotherm was applied to investigate the interactions between the polymers and target compounds: cryptotanshinone; tanshinone I; tanshinone IIA. Thorough comparison revealed that the polymer with a carboxyl group possessed the highest reorganization of the three compounds. After that, the obtained polymer was applied as the sorbent in the solid-phase extraction process to separate the target compounds from methanol extract. The loading volume of extract solution on the sorbent was determined by adsorption isotherm equation and practical test. Under optimized washing and elution conditions, 0.35 mg/g of cryptotanshinone, 0.33 mg/g of tanshinone I, and 0.27 mg/g of tanshinone IIA from plant were obtained by quantitative HPLC analysis. Moreover, six commercial functional drinks containing tanshinones were purified and analyzed.
Keywords: Ionic liquid-modified porous polymer; Molecularly imprinting; Adsorption isotherm; Solid-phase extraction; Tanshinones

Functionalization of the surfaces of silica particles is often the first step in their various applications. An improved heterogeneous Fmoc-Cl fluorescent assay using an aqueous solution was developed to detect the number of amino groups on solid-phase supports. The fluorescent Fmoc-Cl method is 50-fold more sensitive than the current UV assay using an organic solvent. This method, together with the homogeneous fluorescamine and OPA assays, is used to detect amino groups on the silica particle surface. The accuracy and effect factors of these methods were examined and the assays were optimized. The results showed that the amine groups on silica particles can produce stronger fluorescence than small amine molecules in solution, because the porous structure of the particle surface is a more hydrophobic environment. The number of active amino groups that can be conjugated with biomolecules is much less than the total number of amino groups on the silica particle. Compared with physical methods, chemical assays involving direct reaction with amino groups would furnish the closest result to the number of active amino groups on the particle surface. Figure Fluorescence emission of Fmoc-Cl in aqueous solution and linear correlation between intensity and concentration
Keywords: Quantification of amino groups; Nanoparticle; Fmoc-Cl; Fluorescamine; o-phthalaldehyde

Accurate LC-MS analyses for microcystins using per-15N-labeled microcystins by Tomoharu Sano; Hiroo Takagi; Kimiyo Nagano; Masataka Nishikawa; Kunimitsu Kaya (2511-2516).
Per-15N-labeled microcystins were prepared for use as surrogates for accurate liquid chromatography–mass spectrometry analysis. Two strains of Microcystis aeruginosa were cultured in 15NO3-containing TS-15 medium. To change from the incorporation of 14N to 15N into all cell components, cells of Microcystis aeruginosa were precultured in Na15NO3-containing medium for more than 6 months. After mass cultivation of the strains, cells of each strain were harvested and lyophilized. Microcystin variants were extracted from the lyophilized cells and per-15N-labeled microcystin variants were purified using high-performance liquid chromatography and high-performance thin-layer chromatography. The structures of per-15N-labeled microcystin variants were confirmed by their mass spectrometry spectra and NMR spectra. When per-15N-labeled microcystins were used as surrogates for quantitative analysis of these toxins in cyanobacterial cells, excellent accuracy (98–106%) was obtained, with the m/z of M+, [M+1]+, and [M+2]+ of both microcystins and the per-15N-labeled microcystins as surrogates being completely separated. In conclusion, per-15N-labeled microcystins are excellent surrogates for microcystin analysis using liquid chromatography–mass spectrometry.
Keywords: Microcystin; Stable isotope; Surrogate; Liquid chromatography–mass spectrometry; Accurate analysis

A simple and sensitive method was developed using hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry for determination of monosaccharides liberated from marine polysaccharides by acidic hydrolysis. Optimal separation of diastereomeric monosaccharides including hexoses, pentoses, and deoxyhexoses was achieved using an aminopropyl bonded column with mobile phase containing ternary solvents (acetonitrile/methanol/water) in conjunction with MS/MS in SRM mode. Mechanisms for fragmentation of deprotonated monosaccharides with regard to cross-ring cleavage were proposed. Matrix effects from coeluting interferences were observed and isotopic-labeled internal standard was used to compensate for the signal suppression. The method demonstrated excellent instrumental limits of detection (LOD), ranging from 0.7 to 4.2 pg. Method LODs range from 0.9 to 5.1 nM. The proposed method was applied to the analysis of polysaccharides in seawater collected from the open leads of the central Arctic Ocean in the summer of 2008.
Keywords: Hydrophilic interaction liquid chromatography; Tandem mass spectrometry; Monosaccharides; Polysaccharides; Marine microcolloids; Marine gels

A headspace SPME-GC-ECD method suitable for determination of chlorophenols in water samples by Paulo de Morais; Teodor Stoichev; M. Clara P. Basto; Pedro N. Carvalho; M. Teresa S. D. Vasconcelos (2531-2538).
A headspace solid phase microextraction coupled to gas chromatography with electron capture detector (HS-SPME-GC-ECD) method was optimized for the determination of seven chlorophenols (CPs) with different levels of chlorination. This is the first time that HS-SPME-GC-ECD with acetylation of the analytes is used for the simultaneous determination of CPs in water samples. The influence of fibre type, derivatization conditions, salt addition, temperature and time of extraction and temperature of desorption was checked. Possible sources of contamination and analyte losses were considered. The best results were obtained with the polydimethylsiloxane/divinylbenzene fibre, derivatization by acetylation using 100 μL of acetic anhydride and 0.1 g of anhydrous sodium carbonate per 10 mL of sample, salt addition of 100 g L−1 sodium chloride, extraction at 70 °C for 60 min and desorption in the GC injector at 260 °C for 6 min. The limits of detection (LOD) for monochlorophenols were 12 and 122 ng L−1 for 2-chlorophenol and 4-chlorophenol, respectively. For polychlorinated CPs, the LODs were lower than 6 ng L−1, values similar to the existing methods that use SPME with derivatization for CPs determination in water samples. The method is suitable for the determination of CPs in most environmental aqueous samples. Repeatability and reproducibility were less than 16.8% and 11.7%, respectively. The optimized method was successfully applied for the analysis of waters with complex matrices such as river and estuarine water samples.
Keywords: Chlorophenols; Gas chromatography; Solid phase microextraction; Water samples

A sensitive and effective method for simultaneous determination of triazolopyrimidine sulfonamide herbicide residues in soil, water, and wheat was developed using ultra-performance liquid chromatography coupled with tandem mass spectrometry. The four herbicides (pyroxsulam, flumetsulam, metosulam, and diclosulam) were cleaned up with an off-line C18 SPE cartridge and detected by tandem mass spectrometry using an electrospray ionization source in positive mode (ESI+). The determination of the target compounds was achieved in <2.0 min. The limits of detection were below 1 μg kg−1, while the limits of quantification did not exceed 3 μg kg−1 in different matrices. Quantitation was determined from calibration curves of standards containing 0.05–100 μg L−1 with r 2 > 0.997. Recovery studies were conducted at three spiked levels (0.2, 1, and 5 μg kg−1 for water; 5, 10, and 100 μg kg−1 for soil and wheat). The overall average recoveries for this method in water, soil, wheat plants, and seeds at three levels ranged from 75.4% to 106.0%, with relative standard deviations in the range of 2.1–12.5% (n = 5) for all analytes.
Keywords: Residue analysis; Ultra-performance liquid chromatography–mass spectrometry; Soil; Wheat; Water; Triazolopyrimidine sulfonamide

This article presents an analytical method based on solid-phase extraction (SPE) and gas chromatography coupled with mass spectrometry for the simultaneous determination of the most frequently used acidic pharmaceutical residues, ibuprofen, diclofenac, naproxen and ketoprofen (KFN), and phenolic endocrine disruptors, bisphenol (BPA), triclosan (TCS), nonylphenol, nonylphenol monoethoxylate and nonylphenol diethoxylate, in wastewater and sewage sludge samples. In the first phase of the study, each compound has been characterized individually and afterwards in mixture as a trimethylsilyl derivative in order to identify the characteristic ions (m/z ratio) constituting the mass spectrum and to choose the ions for quantification and confirmation. Subsequently, derivatization was evaluated by testing different variables such as the volume of the derivatization solvent bis(trimethylsilyl)trifluoroacetamide and the effect of each catalyst, pyridine and 1% trimethyl chlorosilane, in the derivatized solution. For the analysis of wastewater samples, two commercial SPE cartridges, C18 and Oasis HLB, were compared for their extraction efficiency of the target compounds. The key parameter of extraction procedure included the effect of pH (2.5, 5.3 and 7) of the loading solution. For solid samples, parameters such as the extracted biomass, the volume of the extraction organic solvent and the effect of matrix interferences in chromatographic analysis were evaluated. By using C18 cartridges as purification procedure and ultrasound sonication, satisfactory mean relative recoveries with BPA-d16 and meclofenamic acid as surrogates were obtained ranging from 91% to 117% for wastewater and 84% to 107% for sewage sludge samples. Nine-point calibration of the standard mixture was performed by linear regression analysis with a correlation coefficient >0.99 for all the tested compounds. Limits of detection for the developed methods were established between 0.3 (KFN) and 14.8 (BPA) ng L−1, and 15.0 (TCS) and 32.9 (BPA) ng g−1 for wastewater and sewage sludge, respectively. Application to real samples of the wastewater treatment plant in Athens, the capital of Greece, demonstrated the presence of all tested compounds in most of the samples. Figure An analytical method for the simultaneous trace determination of acidic pharmaceuticals and phenolic endocrine disrupting chemicals in wastewater and sewage sludge by gas chromatography-mass spectrometry
Keywords: Acidic pharmaceuticals; Phenolic endocrine disrupting chemicals; Environmental samples; Silyl derivatization; SPE; GC-MS

Determination of labile inorganic and organic species of Al and Cu in river waters using the diffusive gradients in thin films technique by Paulo Sergio Tonello; Danielle Goveia; André Henrique Rosa; Leonardo Fernandes Fraceto; Amauri Antonio Menegário (2563-2570).
The diffusive gradients in thin films (DGT) technique, using a diffusive gel or a restrictive gel, was evaluated for the determination of labile inorganic and organic species of Al and Cu in model synthetic solutions and river water samples. Experiments were performed both in situ and in the laboratory. In the solutions containing Al ions, the major labile fraction consisted of inorganic species. The organic complex fractions were mainly kinetically inert. For the model Cu solutions, the most labile fraction consisted of inorganic species; however, significant amounts of labile organic complexes of Cu were also present. A comparison was made between the results obtained using restrictive gel DGT and tangential flow ultrafiltration (TF-UF). The Cu fraction determined by restrictive gel DGT (corresponding to the "free" ions plus the labile fraction of small molecular size complexes) was larger than that determined by TF-UF (corresponding to all small molecular size ions), suggesting that the techniques exhibited different porosities for discrimination of inorganic species. For the river water samples analyzed in the laboratory, less than 45% of the analytes were present in labile forms, with most being organic species. For the in situ measurements, the labile inorganic and organic fractions were larger than those obtained in the laboratory analyses. These differences could have been due to errors incurred during sample collection and storage. All results were consistent with those found using two different methods, namely, solid-phase extraction and the DGT technique employing the apparent diffusion coefficient.
Keywords: Diffusive gradients in thin films; Lability; Copper; Aluminum; Speciation

Influence of olive (cv Grignano) fruit ripening and oil extraction under different nitrogen regimes on volatile organic compound emissions studied by PTR-MS technique by Alice Vezzaro; Andrea Boschetti; Rossana Dell’Anna; Roberto Canteri; Mariano Dimauro; Angelo Ramina; Massimo Ferasin; Claudio Giulivo; Benedetto Ruperti (2571-2582).
Volatile organic compounds of extra virgin olive oils obtained from the local Italian cultivar Grignano were measured by proton transfer reaction–mass spectrometry (PTR-MS). Oils were extracted by olives harvested at different ripening stages across veraison, performing each extraction step and the whole extraction process in nitrogen atmosphere to observe the changes in the volatile profiles of the oils. Principal component analysis carried out on the full spectral signature of the PTR-MS measurements showed that the stage of ripening has a stronger effect on the global definition of volatile profiles than the use of nitrogen during oil extraction. The fingerprint-like chemical information provided by the spectra were used to construct a heat map, which allowed the dynamical representation of the multivariate nature of mass evolution during the ripening process. This provided the first evidence that some groups of volatile organic compounds displayed a time course of regulation with coordinated increasing or decreasing trends in association with specific stages of fruit ripening. Figure Influence of olive fruit ripening and oil extraction in nitrogen atmosphere on volatile organic compounds has been evaluated by means of PTR-MS and multivariate data analysis.
Keywords: Olive oil; Olive ripening; Proton transfer reaction–mass spectrometry; Volatile compounds; Principal components analysis; Heat maps