Analytical and Bioanalytical Chemistry (v.405, #19)

Optical nanosensing in cells by Francesco Baldini (6143-6144).
has been senior scientist at the Institute of Applied Physics of the National Council of Research, Italy, since 2001. His research activity has been devoted to the development of optical sensors and to the application of optical methods for restoration of paintings and frescoes. Now his research interests lie in optical fibre sensors and systems for chemical and biochemical applications. Since 2005 he has been member of the International Advisory Board of the journal Analytical and Bioanalytical Chemistry. In 2009 he was nominated fellow of SPIE for his achievements in biological and chemical sensing in biomedicine, and in January 2013 was elected vice-president of the Italian Society of Optics and Photonics.

Evaluation of diverse peptidyl motifs for cellular delivery of semiconductor quantum dots by Kelly Boeneman Gemmill; Markus Muttenthaler; James B. Delehanty; Michael H. Stewart; Kimihiro Susumu; Philip E. Dawson; Igor L. Medintz (6145-6154).
Cell-penetrating peptides (CPPs) have rapidly become a mainstay technology for facilitating the delivery of a wide variety of nanomaterials to cells and tissues. Currently, the library of CPPs to choose from is still limited, with the HIV TAT-derived motif still being the most used. Among the many materials routinely delivered by CPPs, nanoparticles are of particular interest for a plethora of labeling, imaging, sensing, diagnostic, and therapeutic applications. The development of nanoparticle-based technologies for many of these uses will require access to a much larger number of functional peptide motifs that can both facilitate cellular delivery of different types of nanoparticles to cells and be used interchangeably in the presence of other peptides and proteins on the same surface. Here, we evaluate the utility of four peptidyl motifs for their ability to facilitate delivery of luminescent semiconductor quantum dots (QDs) in a model cell culture system. We find that an LAH4 motif, derived from a membrane-inserting antimicrobial peptide, and a chimeric sequence that combines a sweet arrow peptide with a portion originating from the superoxide dismutase enzyme provide effective cellular delivery of QDs. Interestingly, a derivative of the latter sequence lacking just a methyl group was found to be quite inefficient, suggesting that even small changes can have significant functional outcomes. Delivery was effected using 1 h incubation with cells, and fluorescent counterstaining strongly suggests an endosomal uptake process that requires a critical minimum number or ratio of peptides to be displayed on the QD surface. Concomitant cytoviability testing showed that the QD–peptide conjugates are minimally cytotoxic in the model COS-1 cell line tested. Potential applications of these peptides in the context of cellular delivery of nanoparticles and a variety of other (bio)molecules are discussed. Figure ᅟ
Keywords: Cell-penetrating peptide; Quantum dot; Nanoparticle; Cell; Delivery; Endocytosis; Labeling; Nanotechnology; Sweet arrow peptide; Quantum dot; Metal affinity

Field-deployable whole-cell bioluminescent biosensors: so near and yet so far by Elisa Michelini; Luca Cevenini; Maria Maddalena Calabretta; Silvia Spinozzi; Cecilia Camborata; Aldo Roda (6155-6163).
The use of smart supports and bioinspired materials to confine living cells and use them for field-deployable biosensors has recently attracted much attention. In particular, bioluminescent whole-cell biosensors designed to respond to different analytes or classes of analyte have been successfully implemented in portable and cost-effective analytical devices. Significant advances in detection technology, biomaterial science, and genetic engineering of cells have recently been reported. Now the challenge is to move from benchtop traditional cell-based assays to portable biosensing devices. Improvement of the analytical performance of these biosensors depends on the availability of optimized bioluminescent reporters, and promising approaches that go beyond reporter gene technology are emerging. To enable handling of cells as ready-to-use reagents, nature-inspired strategies have been used, with the objective of keeping cells in a dormant state until use. Several issues must still be investigated, for example long-term viability of cells, the possibility of performing real-time analysis, and multiplexing capability. Figure Concept of whole-cell bioluminescent biosensor
Keywords: Whole-cell biosensor; Bioluminescence; Nanomaterial; Cell encapsulation; Analytical device

Plasmonic nanoprobes for intracellular sensing and imaging by Hsiangkuo Yuan; Janna K. Register; Hsin-Neng Wang; Andrew M. Fales; Yang Liu; Tuan Vo-Dinh (6165-6180).
Recent advances in integrating nanotechnology and optical microscopy offer great potential in intracellular applications with improved molecular information and higher resolution. Continuous efforts in designing nanoparticles with strong and tunable plasmon resonance have led to new developments in biosensing and bioimaging, using surface-enhanced Raman scattering and two-photon photoluminescence. We provide an overview of the nanoprobe design updates, such as controlling the nanoparticle shape for optimal plasmon peak position; optical sensing and imaging strategies for intracellular nanoparticle detection; and addressing practical challenges in cellular applications of nanoprobes, including the use of targeting agents and control of nanoparticle aggregation. Figure Plasmonic nanoprobe characterization (TEM, simulation) and applications in pH sensing, SERS mapping, and TPL imaging
Keywords: Plasmonic; Nanoparticle; Surface-enhanced Raman scattering; Two-photon photoluminescence; Biosensing; Cellular imaging

Oligonucleotide optical switches for intracellular sensing by A. Giannetti; S. Tombelli; F. Baldini (6181-6196).
Fluorescence imaging coupled with nanotechnology is making possible the development of powerful tools in the biological field for applications such as cellular imaging and intracellular messenger RNA monitoring and detection. The delivery of fluorescent probes into cells and tissues is currently receiving growing interest because such molecules, often coupled to nanodimensional materials, can conveniently allow the preparation of small tools to spy on cellular mechanisms with high specificity and sensitivity. The purpose of this review is to provide an exhaustive overview of current research in oligonucleotide optical switches for intracellular sensing with a focus on the engineering methods adopted for these oligonucleotides and the more recent and fascinating techniques for their internalization into living cells. Oligonucleotide optical switches can be defined as specifically designed short nucleic acid molecules capable of turning on or modifying their light emission on molecular interaction with well-defined molecular targets. Molecular beacons, aptamer beacons, hybrid molecular probes, and simpler linear oligonucleotide switches are the most promising optical nanosensors proposed in recent years. The intracellular targets which have been considered for sensing are a plethora of messenger-RNA-expressing cellular proteins and enzymes, or, directly, proteins or small molecules in the case of sensing through aptamer-based switches. Engineering methods, including modification of the oligonucleotide itself with locked nucleic acids, peptide nucleic acids, or l-DNA nucleotides, have been proposed to enhance the stability of nucleases and to prevent false-negative and high background optical signals. Conventional delivery techniques are treated here together with more innovative methods based on the coupling of the switches with nano-objects.
Keywords: Intracellular sensing; Molecular beacon; Aptamer; Fluorescence; Living cells; Intracellular delivery

A photoinduced electron transfer-based nanoprobe as a marker of acidic organelles in mammalian cells by María J. Marín; Francisco Galindo; Paul Thomas; Tom Wileman; David A. Russell (6197-6207).
Photoinduced electron transfer (PET)-based molecular probes have been successfully used for the intracellular imaging of the pH of acidic organelles. In this study, we describe the synthesis and characterization of a novel PET-based pH nanoprobe and its biological application for the signaling of acidic organelles in mammalian cells. A fluorescent ligand sensitive to pH via the PET mechanism that incorporates a thiolated moiety was synthesized and used to stabilize gold nanoparticles (2.4 ± 0.6 nm), yielding a PET-based nanoprobe. The PET nanoprobe was unambiguously characterized by transmission electron microscopy, proton nuclear magnetic resonance, Fourier transform infrared, ultraviolet-visible absorption, and steady-state/time-resolved fluorescence spectroscopies which confirmed the functionalization of the gold nanoparticles with the PET-based ligand. Following a classic PET behavior, the fluorescence emission of the PET-based nanoprobe was quenched in alkaline conditions and enhanced in an acidic environment. The PET-based nanoprobe was used for the intracellular imaging of acidic environments within Chinese hamster ovary cells by confocal laser scanning microscopy. The internalization of the nanoparticles by the cells was confirmed by confocal fluorescence images and also by recording the fluorescence emission spectra of the intracellular PET-based nanoprobe from within the cells. Co-localization experiments using a marker of acidic organelles, LysoTracker Red DND-99, and a marker of autophagosomes, GFP-LC3, confirm that the PET-based nanoprobe acts as marker of acidic organelles and autophagosomes within mammalian cells. Figure A PET based ligand has been used to functionalize gold nanoparticles to develop a pH sensitive nanoprobe. The fluorescence of the nanoprobe, following the PET mechanism, is enhanced in acidic environments and quenched at neutral pH. A combination of spectroscopy and confocal fluorescence microscopy is used for confirmation of the cellular uptake of the nanoprobe by Chinese hamster ovary cells. The PET-based nanoprobe has been used as a marker of acidic organelles and autophagosomes within the CHO cells
Keywords: Gold nanoparticles; Fluorescence; Intracellular analysis; pH sensing; Autophagosomes

Intracellular SERS hybrid probes using BSA–reporter conjugates by Andrea Hornemann; Daniela Drescher; Sabine Flemig; Janina Kneipp (6209-6222).
Surface-enhanced Raman scattering (SERS) hybrid probes are characterized by the typical spectrum of a reporter molecule. In addition, they deliver information from their biological environment. Here, we report SERS hybrid probes generated by conjugating different reporter molecules to bovine serum albumin (BSA) and using gold nanoparticles as plasmonic core. Advantages of the BSA-conjugate hybrid nanoprobes over other SERS nanoprobes are a high biocompatibility, stabilization of the gold nanoparticles in the biological environment, stable reporter signals, and easy preparation. The coupling efficiencies of the BSA–reporter conjugates were determined by MALDI-TOF-MS. The conjugates’ characteristic SERS spectra differ from the spectra of unbound reporter molecules. This is a consequence of the covalent coupling, which leads to altered SERS enhancement and changes in the chemical structures of the reporter and of BSA. The application of the BSA–reporter conjugate hybrid probes in 3T3 cells, including duplex imaging, is demonstrated. Hierarchical cluster analysis and principal components analysis were applied for multivariate imaging using the SERS signatures of the incorporated SERS hybrid nanoprobes along with the spectral information from biomolecules in endosomal structures of cells. The results suggest more successful applications of the SERS hybrid probes in cellular imaging and other unordered high-density bioanalytical sensing. Figure Single pixel spectrum obtained with SERS hybrid nanoprobes (here: BSA-AO conjugate on gold nanoparticles) inside living 3T3 cells. The distribution of SERS hybrid nanoprobes in 3T3 fibroblast cells can be obtained from chemical mapping, and from hierarchical cluster analysis (HCA) mapping employing the full spectral range from 300–1700 cm-1
Keywords: Bovine serum albumin; Nanosensor; SERS multiplexing; 3T3 cells; Gold nanoparticles; Hybrid nanoprobe

Imaging intracellular viscosity by a new molecular rotor suitable for phasor analysis of fluorescence lifetime by Antonella Battisti; Silvio Panettieri; Gerardo Abbandonato; Emanuela Jacchetti; Francesco Cardarelli; Giovanni Signore; Fabio Beltram; Ranieri Bizzarri (6223-6233).
The arsenal of fluorescent probes tailored to functional imaging of cells is rapidly growing and benefits from recent developments in imaging strategies. Here, we present a new molecular rotor, which displays strong absorption in the green region of the spectrum, very little solvatochromism, and strong emission sensitivity to local viscosity. The emission increase is paralleled by an increase in emission lifetime. Owing to its concentration-independent nature, fluorescence lifetime is particularly suitable to image environmental properties, such as viscosity, at the intracellular level. Accordingly, we demonstrate that intracellular viscosity measurements can be efficiently carried out by lifetime imaging with our probe and phasor analysis, an efficient method for measuring lifetime-related properties (e.g., bionalyte concentration or local physicochemical features) in living cells. Notably, we show that it is possible to monitor the partition of our probe into different intracellular regions/organelles and to follow mitochondrial de-energization upon oxidative stress.
Keywords: Intracellular viscosity; Molecular rotor; Phasor approach; FLIM; Mitochondria

is Assistant Professor of Analytical Chemistry at the University of Isfahan, in Isfahan (Iran), where he has been a member of the faculty since 2011. He received his B.S. degree from Ferdowsi University of Mashhad, in Mashhad (Iran), and his M.S. and Ph.D. degrees from Sharif University of Technology, in Tehran (Iran). His research spans a range of areas in analytical chemistry, including chemometrics, multivariate techniques, “hyphenated” and multidimensional chromatographic techniques, and medicinal plant analysis research. He spent his sabbatical with Romà Tauler at the Institute of Environmental Assessment and Water Research (IDÆA), Spanish National Research Council (CSIC), in Barcelona (Spain). graduated from the University of Novi Sad, Serbia. In 2009 he was awarded a predoctoral fellowship from the Spanish National Research Council (CSIC) to work on his thesis at the Institute of Environmental Assessment and Water Research (IDÆA) in Barcelona. His research is focused on marine oil pollution, geochemistry of petroleum, and the environmental fate and effects of petroleum, and involves the application of comprehensive two-dimensional gas chromatography and effect-directed analysis. received his B.Sc. degree in chemistry and his Ph.D. degree in chemistry in 1985 from the Autonomous University of Barcelona. He is Research Professor at the Institute of Environmental Assessment and Water Research (IDÆA), Spanish National Research Council (CSIC), in Barcelona (Spain). His main research interests are in the field of environmental chemistry, focusing on the transformation processes and the pathway of organic contaminants in the environment and engineered ecosystems. He was an editor of the Analytical Chemistry A pages (2003–2005) and is member of the editorial board of the International Journal of Environmental Chemistry. is Research Professor at the Institute of Environmental Assessment and Water Research (IDÆA), Spanish National Research Council (CSIC), in Barcelona (Spain). He is Editor in Chief of Chemometrics and Intelligent Laboratory Systems. He is a recipient of many awards, including the 2012 ERC Advanced Grant award, the 2009 Eastern Analytical Symposium Award for Achievements in Chemometrics, and the 2009 Kowalski Prize from the Journal of Chemometrics. He is also President of the Catalan Chemistry Society. His main research interests are in chemometrics, especially the development of multivariate curve resolution methods for the analysis of multiway and multiset data, and their applications to environmental chemistry, omic sciences, analytical and bioanalytical chemistry and solution chemistry. Multivariate curve resolution–alternating least squares (MCR–ALS) analysis is proposed to solve chromatographic challenges during two-dimensional gas chromatography–time-of-flight mass spectrometry (GC × GC–TOFMS) analysis of complex samples, such as crude oil extract. In view of the fact that the MCR–ALS method is based on the fulfillment of the bilinear model assumption, three-way and four-way GC × GC–TOFMS data are preferably arranged in a column-wise superaugmented data matrix in which mass-to-charge ratios (m/z) are in its columns and the elution times in the second and first chromatographic columns are in its rows. Since m/z values are common for all measured spectra in all second-column modulations, unavoidable chromatographic challenges such as retention time shifts within and between GC × GC–TOFMS experiments are properly handled. In addition, baseline/background contributions can be modeled by adding extra components to the MCR–ALS model. Another outstanding aspect of MCR–ALS analysis is its extreme flexibility to consider all samples (standards, unknowns, and replicates) in a single superaugmented data matrix, allowing joint analysis. In this way, resolution, identification, and quantification results can be simultaneously obtained in a very fast and reliable way. The potential of MCR–ALS analysis is demonstrated in GC × GC–TOFMS analysis of a North Sea crude oil extract sample with relative errors in estimated concentrations of target compounds below 6.0 % and relative standard deviations lower than 7.0 %. The results obtained, along with reasonable values for the lack of fit of the MCR–ALS model and high values of the reversed match factor in mass spectra similarity searches, confirm the reliability of the proposed strategy for GC × GC–TOFMS data analysis.
Keywords: Comprehensive two-dimensional gas chromatography; Multivariate curve resolution; Multivariate curve resolution–alternating least squares; Crude oil; Time-of-flight mass spectrometry

In this work, we highlight the size-independent influence of the material properties of nanoparticles (NPs) on their retention behavior in asymmetric-flow field-flow fractionation (A4F) by comparing four NP populations with similar nominal size. The phenomena described here suggest there are limits to the effectiveness and accuracy of using a single type of NP standard (polystyrene beads most typically) in order to generically calibrate retention time in normal mode elution. The dual objectives of this paper are to (1) demonstrate the uncertainties resulting from current practice and (2) initiate a discussion of these effects and their origins. The results presented here illustrate clearly that the retention time is higher for metallic NPs relative to lower (bulk) density NPs. By modifying the fundamental field-flow fractionation equation to account for differences in particle density, we show that the effect of the gravitational force is finite but insignificant for NPs. We postulate that the observed material-dependent retention behavior may be attributed to differences in the attractive van der Waals force between the NPs and the accumulation wall (membrane surface). We hope that our results will stimulate discussion and reassessment of the calibration procedure, perhaps by more fully accounting for all influential material parameters relevant to the fractionation of nanoscale particles by A4F.
Keywords: Field-flow fractionation; Nanoparticles/nanotechnology; Separations/theory

Surface modification by means of wear protective and antibacterial coatings represents, nowadays, a crucial challenge in the biomaterials field in order to enhance the lifetime of bio-devices. It is possible to tailor the properties of the material by using an appropriate combination of high wear resistance (e.g., nitride or carbide coatings) and biocide agents (e.g., noble metals as silver) to fulfill its final application. This behavior is controlled at last by the outmost surface of the coating. Therefore, the analytical characterization of these new materials requires high-resolution analytical techniques able to provide information about surface and depth composition down to the nanometric level. Among these techniques are Rutherford backscattering spectrometry (RBS), glow discharge optical emission spectroscopy (GDOES), and angle resolved X-ray photoelectron spectroscopy (ARXPS). In this work, we present a comparative RBS–GDOES–ARXPS study of the surface characterization of Ag–TiCN coatings with Ag/Ti atomic ratios varying from 0 to 1.49, deposited at room temperature and 200 °C. RBS analysis allowed a precise quantification of the silver content along the coating with a non-uniform Ag depth distribution for the samples with higher Ag content. GDOES surface profiling revealed that the samples with higher Ag content as well as the samples deposited at 200 °C showed an ultrathin (1–10 nm) Ag-rich layer on the coating surface followed by a silver depletion zone (20–30 nm), being the thickness of both layers enhanced with Ag content and deposition temperature. ARXPS analysis confirmed these observations after applying general algorithm involving regularization in addition to singular value decomposition techniques to obtain the concentration depth profiles. Finally, ARXPS measurements were used to provide further information on the surface morphology of the samples obtaining an excellent agreement with SEM observations when a growth model of silver islands with a height d = 1.5 nm and coverage θ = 0.20 was applied to the sample with Ag/Ti = 1.49 and deposited at room temperature. Figure SEM micrograph of silver nanocluster surface segregation on bioactive AgTiCN coatings as analyzed by a) GDOES, b) RBS, and c) ARXPS depth profiles
Keywords: Ag–TiCN; Surface characterization; SEM; RBS; GDOES; ARXPS

Actinomycin D (Act D), an oncogenic c-Myc promoter binder, interferes with the action of RNA polymerase. There is great demand for high-throughput technology able to monitor the activity of DNA-binding drugs. To this end, binding of 7-aminoactinomycin D (7AAD) to the duplex c-Myc promoter was investigated by use of 2D-photoluminescence emission (2D-PLE), and the resulting data were subjected to analysis by use of convenient and powerful multi-way approaches. Fluorescence measurements were performed by use of the quantum dot (QD)-conjugated c-Myc promoter. Intercalation of 7AAD within duplex base pairs resulted in efficient energy transfer from drug to QD via fluorescence resonance energy transfer (FRET). Multi-way analysis of the three-way data array obtained from titration experiments was performed by use of restricted Tucker3 and hard trilinear decomposition (HTD). These techniques enable analysis of high-dimensional and complex data from nanobiological systems which include several spectrally overlapped structures. It was almost impossible to obtain robust and meaningful information about the FRET process for such high overlap data by use of classical analysis. The soft approach had the important advantage over univariate classical methods of enabling us to investigate the source of variance in the fluorescence signal of the DNA–drug complex. It was established that hard trilinear decomposition analysis of FRET-measured data overcomes the problem of rank deficiency, enabling calculation of concentration profiles and pure spectra for all species, including non-fluorophores. The hard modeling approach was also used for determination of equilibrium constants for the hybridization and intercalation equilibria, using nonlinear fit data analysis. The intercalation constant 3.6 × 106 mol−1 L and hybridization stability 1.0 × 108 mol−1 L obtained were in good agreement with values reported in the literature. The analytical concentration of the QD-labeled DNA was determined by use of nonlinear fitting, without using external standard calibration samples. This study was a successful application of multi-way chemometric methods to investigation of nano-biotechnological systems where several overlapped species coexist in solution. Figure ᅟ
Keywords: Quantum dot; FRET; Multi-way analysis; Actinomycin D; Drug–DNA interaction

Fluorescent sensing ochratoxin A with single fluorophore-labeled aptamer by Qiang Zhao; Xia Geng; Hailin Wang (6281-6286).
We explored a fluorescent strategy for sensing ochratoxin A (OTA) by using a single fluorophore-labeled aptamer for detection of OTA. This method relied on the change of the fluorescence intensity of the labeled dye induced by the specific binding of the fluorescent aptamer to OTA. Different fluorescein labeling sites of aptamers were screened, including the internal thymine bases, 3′-end, and 5′-end of the aptamer, and the effect of the labeling on the aptamer affinity was investigated. Some fluorophore-labeled aptamers showed a signal-on or signal-off response. With the fluorescent aptamer switch, simple, rapid, and selective sensing of OTA at nanomolar concentrations was achieved. OTA spiked in diluted red wine could be detected, showing the feasibility of the fluorescent aptamer for a complex matrix. This method shows potential for designing aptamer sensors for other targets. Figure A simple fluorescent approach for OTA sensing is achieved by using single fluorophore-labeled aptamer. A fluorophore is attached on one site of the aptamer. The affinity binding of OTA induces the alteration of fluorescence properties of the labeled fluorophore as the consequence of the conformation change of the aptamer. OTA can be detected by measuring the change of fluorescence signals of the labeled dye
Keywords: Aptamer; Ochratoxin A; Fluorescence; Sensor; Labeling

A sensitive and robust liquid chromatography–tandem mass spectrometry method allowing the rapid screening and confirmation of ten synthetic corticosteroids in bovine and porcine muscle tissue was developed and validated. The validation was conducted according to Commission Decision 2002/657/EC, Sect. 3.1.3 (“Validation according to alternative models”), by applying a matrix-comprehensive in-house validation concept. The decision limit, detection capability, recovery, repeatability, within-laboratory-reproducibility and measurement uncertainty were calculated. Furthermore, a factorial effect analysis was conducted to identify factors that have a significant influence on the method. To this end, factors considered to be relevant for the method in routine analysis (e.g. operator, duration of storage of the extracts before measurement, different lots of the cartridges and different species) were systematically varied on two levels during the validation study. Subsequently, the extent to which these factors influence the measurement results of the individual analytes was examined.
Keywords: Corticosteroids; Bovine and porcine muscle; Liquid chromatography–tandem mass spectrometry; Matrix-comprehensive in-house validation; Factorial effect analysis; Residue control

Determination of cocaine and metabolites in hair by column-switching LC-MS-MS analysis by Marcela Nogueira Rabelo Alves; Gabriele Zanchetti; Alberto Piccinotti; Silvia Tameni; Bruno Spinosa De Martinis; Aldo Polettini (6299-6306).
A method for rapid, selective, and robust determination of cocaine (CO) and metabolites in 5-mg hair samples was developed and fully validated using a column-switching liquid chromatography–tandem mass spectrometry system (LC-MS-MS). Hair samples were decontaminated, segmented, incubated overnight in diluted HCl, and centrifuged, and the diluted (1:10 with distilled water) extracts were analyzed in positive ionization mode monitoring two reactions per analyte. Quantifier transitions were: m/z 304.2→182.2 for CO, m/z 290.1→168.1 for benzoylecgonine (BE), and m/z 318.2→196.2 for cocaethylene (CE). The lower limit of quantification (LLOQ) was set at 0.05 ng/mg for CO and CE, and 0.012 ng/mg for BE. Imprecision and inaccuracy at LLOQ were lower than 20 % for all analytes. Linearity ranged between 0.05 and 50.0 ng/mg for CO and CE and 0.012 and 12.50 ng/mg for BE. Selectivity, matrix effect, process efficiency, recovery, carryover, cross talk, and autosampler stability were also evaluated during validation. Eighteen real hair samples and five samples from a commercial proficiency testing program were comparatively examined with the proposed multidimensional chromatography coupled with tandem mass spectrometry procedure and our reference gas chromatography coupled to mass spectrometry (GC-MS) method. Compared with our reference GC-MS method, column-switching technique and the high sensitivity of the tandem mass spectrometry detection system allowed to significantly reduce sample amount (×10) with increased sensitivity (×2) and sample throughput (×4), to simplify sample preparation, and to avoid that interfering compounds and ions impaired the ionization and detection of the analytes and deteriorate the performance of the ion source.
Keywords: Column switching; LC-MS-MS; Hair testing; Cocaine and metabolites; Drugs of abuse

Ketamine-derived designer drug methoxetamine: metabolism including isoenzyme kinetics and toxicological detectability using GC-MS and LC-(HR-)MS n by Markus R. Meyer; Martina Bach; Jessica Welter; Michael Bovens; Alain Turcant; Hans H. Maurer (6307-6321).
Methoxetamine (MXE; 2-(3-methoxyphenyl)-2-(N-ethylamino)-cyclohexanone), a ketamine analog, is a new designer drug and synthesized for its longer lasting and favorable pharmacological effects over ketamine. The aims of the presented study were to identify the phases I and II metabolites of MXE in rat and human urine by GC-MS and LC-high-resolution (HR)-MS n and to evaluate their detectability by GC-MS and LC-MS n using authors’ standard urine screening approaches (SUSAs). Furthermore, human cytochrome P450 (CYP) enzymes were identified to be involved in the initial metabolic steps of MXE in vitro, and respective enzyme kinetic studies using the metabolite formation and substrate depletion approach were conducted. Finally, human urine samples from forensic cases, where the ingestion of MXE was suspected, were analyzed. Eight metabolites were identified in rat and different human urines allowing postulation of the following metabolic pathways: N-deethylation, O-demethylation, hydroxylation, and combinations as well as glucuronidation or sulfation. The enzyme kinetic studies showed that the initial metabolic step in humans, the N-deethylation, was catalyzed by CYP2B6 and CYP3A4. Both SUSAs using GC-MS or LC-MS n allowed monitoring an MXE intake in urine.
Keywords: Designer drugs; Methoxetamine; MXE; Cytochrome P450; GC-MS; LC-MS

A new, sensitive and fast method for the simultaneous determination of pyrazinamide, isoniazid, streptomycin, ethambutol, and rifampicin in human plasma was developed and validated. The method required only 100 μL of plasma and one step for sample preparation by protein precipitation. The drugs were separated by using a hydrophilic interaction liquid chromatography (HILIC) column. The mobile phase was methanol and water (0.1 % formic acid and 5 mM ammonium acetate, pH 3.0 ± 0.1) in a ratio of 65:35 (v/v), which was eluted at an isocratic flow rate of 0.5 mL/min. Tandem mass spectrometry was performed with a triple-quadrupole tandem mass spectrometer. By use of the HILIC column, the detection was free of ion-pair reagents in the mobile phase, with no significant matrix effects. The total run time was less than 2 min for each sample. The method was validated by evaluating its selectivity, sensitivity, linearity, accuracy, and precision according to US Food and Drug Administration guidelines. The lower limit of quantification was 4.0 ng/mL for pyrazinamide, isoniazid, and rifampicin, 0.5 ng/mL for ethambutol, and 10.0 ng/mL for streptomycin. The intraday precision and interday precision were less than 9 %, with the accuracy ranging between −9.3 and 7.3 %. The method was successfully applied to therapeutic drug monitoring of 33 patients with tuberculosis after administration of standard antituberculosis drugs. The method has been proved to meet the high-throughput requirements in therapeutic drug monitoring. Scatter plots of 2-h plasma drug concentration of patients after receiving a standard medication. (The lower line and the upper line represent the low and high levels of the expected plasma concentrations of the antituberculosis drugs in tuberculosis patients. Open triangles female, age 0–20 years; closed triangles male, age 0–20 years; open diamonds female, age 21–40 years; closed diamonds male, age 21–40 years; open squares female, age 41–60 years; closed squares male, age 41–60; open circles female, age 61–80 years; closed circles male, age 61–80 years)
Keywords: Antituberculosis drug; Therapeutic drug monitoring; Hydrophilic interaction liquid chromatography; Tandem mass spectrometry

A reliable quantification by LC-ESI-MS/MS as the most suitable analytical method for polar substances in the aquatic environment is usually hampered by matrix effects from co-eluting compounds, which are unavoidably present in environmental samples. The standard addition method (SAM) is the most appropriate method to compensate matrix effects. However, when performed manually, this method is too labour- and time-intensive for routine analysis. In the present work, a fully automated SAM using a multi-purpose sample manager “Open Architecture UPLC®-MS/MS” (ultra-performance liquid chromatography tandem mass spectrometry) was developed for the sensitive and reliable determination of 29 polar pesticide metabolites in environmental samples. A four-point SAM was conducted parallel to direct-injection UPLC-ESI-MS/MS determination that was followed by a work flow to calculate the analyte concentrations including monitoring of required quality criteria. Several parameters regarding the SAM, chromatography and mass spectrometry conditions were optimised in order to obtain a fast as well as reliable analytical method. The matrix effects were examined by comparison of the SAM with an external calibration method. The accuracy of the SAM was investigated by recovery tests in samples of different catchment areas. The method detection limit was estimated to be between 1 and 10 ng/L for all metabolites by direct injection of a 10-μL sample. The relative standard deviation values were between 2 and 10 % at the end of calibration range (30 ng/L). About 200 samples from different water bodies were examined with this method in the Rhine and Ruhr region of North Rhine-Westphalia (Germany). Approximately 94 % of the analysed samples contained measurable amounts of metabolites. For most metabolites, low concentrations ≤0.10 μg/L were determined. Only for three metabolites were the concentrations in ground water significantly higher (up to 20 μg/L). In none of the examined drinking water samples were the health-related indication values (between 1 and 3 μg/L) for non-relevant metabolites exceeded.
Keywords: Pesticide metabolites; Non-relevant metabolites; LC-MS/MS; Ion suppression; Matrix effects; Automated standard addition method (SAM)

In this work, molecularly imprinted polymer (MIP) particles were synthesized using a semicovalent method based on a specific thermally reversible bond, and these particles were used for the rapid detection of the azo dye acid orange II. The imprinted polymers—which were prepared via the covalent reaction of 3-(triethoxysilyl)propyl isocyanate with the template molecules—were attached to the surface of silica-coated magnetic nanoparticles, and a simple thermal reaction was then performed to remove the templates, leaving spaces with specific noncovalent bonds for target re-recognition. The conditions for the synthesis of the MIP were optimized during the polymerization experiments to improve the adsorption capacity and selectivity. The resulting polymers were characterized using Fourier transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy to confirm their structure. The MIPs were subjected to an online solid-phase extraction and a magnetic molecularly imprinted solid-phase extraction procedure. For both methods, all samples were prepared with spiking levels of 5.0, 10.0, and 15.0 μg kg-1 using high-performance liquid chromatography with UV/vis detection; after the preconcentration of 50-mL sample solutions, the enhancement factors reached 710 and 629. The limits of detection (signal-to-noise ratio of 3) were 9.83 and 17.41 ng L-1, with relative standard deviations (n = 9) of 6.72 % and 8.25 %, respectively, for a 4.0 μg L−1 standard template solution. These two methods were developed to quantify trace acid orange II contents in food and environmental samples; the recoveries ranged from 72 to 105 % and from 70 to 94 %, respectively.
Keywords: Acid orange II molecularly imprinted polymer; Semicovalent; Solid-phase extraction; Food and environmental samples

Identification of animal glue and hen-egg yolk in paintings by use of enzyme-linked immunosorbent assay (ELISA) by M. Palmieri; M. Vagnini; L. Pitzurra; B. G. Brunetti; L. Cartechini (6365-6371).
We report the development of an indirect ELISA procedure for specific identification of chicken-egg yolk and animal glues in painting micro-samples. The results presented integrate previously published work on ELISA recognition of bovine β-casein and chicken ovalbumin in painting materials. The integrated final ELISA procedure—optimised for protein extraction, immuno-reagent concentrations, blocking solution, incubation time, and temperature—enables multiplex identification, in single samples, of proteinaceous materials, i.e. chicken-egg yolk and albumen, animal glues, and bovine milk and/or casein, mainly used by painters in the past. The procedure has been systematically tested on laboratory models of mural and easel paintings, both naturally and artificially aged, to assess possible inhibitory effects on the immuno-reaction caused by inorganic painting materials (pigments and substrates) and by protein degradation resulting from aging processes. Real samples from case studies, which had previously been investigated and characterised by spectroscopy and chromatography, were successfully studied by use of the developed ELISA procedure. The commercial availability of all the immuno-reagents used, the affordable analytical equipment, and the specificity, sensitivity, and rapidity of ELISA make this method very attractive to diagnostic laboratories in the field of cultural heritage science. Possible further developments to the analytical potential of this technique include improvement of antibody performance and inclusion of other classes of bio-molecules as analytical targets. Figure An ELISA indirect procedure is reported for the specific identification of chicken egg-yolk and animal glues in micro-samples from historical paintings; the method was experimented on laboratory models of mural and easel paintings, both naturally and artificially aged
Keywords: ELISA; Paintings; Proteinaceous binder; Animal glue; Egg yolk

Oxygen consumption by conserved archaeological wood by Martin N. Mortensen; Henning Matthiesen (6373-6377).
Rates of oxygen consumption have been measured over extended time periods for 29 whole samples of conserved, archaeological wood and four samples of fresh, unconserved wood, at 50 % relative humidity and room temperature. Samples from the Swedish Warship Vasa and the Danish Skuldelev Viking ships are included. Most rates were close to 1 μg O2 (g wood)−1 day−1 and the process persisted for several years at least. Consumption of oxygen is related to change in chemical composition, which is, in turn, related to degradation. It is thus demonstrated that despite conservation, waterlogged archaeological wood continues to degrade in a museum climate. Figure The warship Vasa in Stockholm. Photo: Hans Hammarskiöld and Swedish National Maritime Museums
Keywords: Real-time aging; Archaeological waterlogged wood; Oxygen consumption rate; Degradation; Vasa; Skuldelev ships

Using UV-absorbance of intrinsic dithiothreitol (DTT) during RP-HPLC as a measure of experimental redox potential in vitro by Angie Seo; Janelle L. Jackson; Jolene V. Schuster; Didem Vardar-Ulu (6379-6384).
Many in-vitro experiments performed to study the response of thiol-containing proteins to changes in environmental redox potentials use dithiothreitol (DTT) to maintain a preset redox environment throughout the experiments. However, the gradual oxidation of DTT during the course of the experiments, and the interaction between DTT and other components in the system, can significantly alter the initial redox potential and complicate data interpretation. Having an internal reporter of the actual redox potential of the assayed sample facilitates direct correlation of biochemical findings with experimental redox status. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used, well-established tool for analysis and purification of biomolecules, including proteins and peptides. Here, we describe a simple, robust, and quantitative RP-HPLC method we developed and tested for determination of the experimental redox potential of an in-vitro sample at the time of the experiment. It exploits the specific UV-absorbance of the oxidized intrinsic DTT in the samples and retains the high resolving power and high sensitivity of RP-HPLC with UV detection.
Keywords: Reversed-phase high-performance liquid chromatography (RP-HPLC); Dithiothreitol (DTT); Redox potential; Nernst equation; Disulfide bond; UV-absorbance