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Analytical and Bioanalytical Chemistry (v.404, #8)

The Olympic Games challenge by Reinhard Meusinger (pp. 2113-2116).

Solution to standard addition challenge by Olaf Rienitz; Anna-Lisa Hauswaldt; Reinhard Jährling (pp. 2117-2118).

Manfred Reichenbächer and Jürgen W. Einax: Challenges in analytical quality assurance by Karl Molt (pp. 2119-2120).

Virginia Litwin and Philip Marder (Eds.): flow cytometry in drug discovery and development by Sang-Ryoul Park (pp. 2121-2122).

2013 Bunsen–Kirchhoff Award for Analytical Spectroscopy by Detlef Günther (pp. 2123-2123).

Elemental speciation by Thorsten Hoffmann; Klaus G. Heumann (pp. 2125-2126).

has been Professor of Analytical Chemistry at Johannes Gutenberg University Mainz since 2003. Before he held a stand-in professorship at the University of Leipzig and was coordinator of the research areas ‘Analytical Methods in the Life Sciences’ and ‘Atmospheric Trace Constituents’ at the Leibniz-Institut für Analytische Wissenschaften – ISAS in Dortmund. His main research activities are the design and development of tailored methods focussing on the chemical characterization of biologically and environmentally relevant matrices. The results are aimed at understanding the interactions between the biosphere and the atmosphere, such as formation and characterization of aerosols from natural or anthropogenic sources and the development of trace analytical techniques to investigate climate archives. Most of the analytical research is focussed on organic analytes in combination with mass spectrometry (gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry) with a certain emphasis on the development of real-time methods for aerosol characterization (aerosol mass spectrometry).is Emeritus Professor of Analytical Chemistry at Johannes Gutenberg University Mainz. His research interests lie in the development and application of analytical methods for the determination of trace elements and trace amounts of elemental species, using inductively coupled plasma mass spectrometry, thermal ionization mass spectrometry, different types of optical atomic spectrometry and electroanalysis as detection methods, and high-performance liquid chromatography, capillary electrophoresis and capillary gas chromatography as separation methods. He has received several awards, including the Clemens Winkler Medal of the German Society for Analytical Chemistry in 2004 for his scientific lifework and for continuous support of analytical chemistry in Germany. In 2007 he received the European Award for Plasma Spectrochemistry. He is a member of several national and international scientific societies, and he was a member of the IUPAC Commission on Atomic Weights and Isotopic Abundances for 12 years and was chairman from 1991 to 1995.

Speciation analysis in on-line aerosol mass spectrometry by Frank Drewnick (pp. 2127-2131).

Ambient aerosol particles affect both the earth’s climate and human health. Both effects depend on the chemical composition of the particles including the binding state of specific elements. Modern on-line aerosol mass spectrometry is capable of measuring aerosol composition with high temporal resolution, avoiding artifacts often introduced by classical off-line methods. The two most common types of on-line aerosol mass spectrometers, laser desorption/ionization mass spectrometers (LDI–MS) and thermal desorption electron impact ionization mass spectrometers (TD–EI-MS) provide reliable information on the most common ambient inorganic and organic aerosol species with high temporal resolution. However, for less common aerosol species identification with both types of instrument is frequently associated with large uncertainties. Here, we provide an overview of the element speciation capabilities of current on-line aerosol mass spectrometry for both carbonaceous and non-carbon-containing aerosol species. We describe limitations and other issues for this type of on-line aerosol analysis.

Keywords: Element speciation; Aerosol; On-line aerosol mass spectrometry; Thermal-desorption electron-impact ionization mass spectrometry; Laser-desorption/ionization mass spectrometry

Speciation of Gd-based MRI contrast agents and potential products of transmetalation with iron ions or parenteral iron supplements by Lena Telgmann; Christoph A. Wehe; Jens Künnemeyer; Ann-Christin Bülter; Michael Sperling; Uwe Karst (pp. 2133-2141).

The risk of transmetalation reactions between gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI) and iron ions is examined under physiological conditions. A fast separation of gadopentetate (Gd-DTPA) and gadoterate (Gd-DOTA) and the respective Fe transmetalation products was accomplished by high-performance liquid chromatography. For detection, the LC system was coupled to an Orbitrap electrospray ionization mass spectrometer to achieve a detection limit as low as 50 nmol/L for Fe-DTPA. In vitro experiments revealed the formation of Fe-DTPA in blood plasma samples with Gd-DTPA and Fe(III) citrate. Analysis after different incubation times of the sample showed that the exchange of the metal ions is significantly dependent on time. If this reaction takes place in the body of MRI patients, this could explain why the disease nephrogenic systemic fibrosis (NSF) develops only after a longer retention of the linear Gd complex in the patient’s body. Transmetalation either with endogenous Fe(II)/Fe(III) ions or with parenteral Fe supplements with Gd-DTPA could not be proven under the applied conditions. The high stability of Gd-DOTA is responsible that transmetalation between this macrocylic complex and neither of the Fe species was observed. These findings are important because NSF only develops after administration of Gd complexes with linear ligands. The results indicate that transmetalation reactions may be a trigger for the development of NSF, if free Fe(III) ions are accessible during a prolonged dwell time of Gd complexes with linear ligands in the patient’s body.

Keywords: Gadolinium; Contrast agent; Magnetic resonance imaging (MRI); Nephrogenic systemic fibrosis (NSF); Transmetalation; Speciation; Hyphenated techniques; Electrospray ionization mass spectrometry; Human blood plasma

Sensitive redox speciation of neptunium by CE–ICP–MS by Nils Stöbener; Samer Amayri; Aaron Gehl; Ugras Kaplan; Kurtis Malecha; Tobias Reich (pp. 2143-2150).

Capillary electrophoresis (CE) was used to separate the neptunium oxidation states Np(IV) and Np(V), which are the only oxidation states of Np that are stable under environmental conditions. The CE setup was coupled to an inductively coupled plasma mass spectrometer (Agilent 7500ce) using a Mira Mist CE nebulizer and a Scott-type spray chamber. The combination of the separation capacity of CE with the detection sensitivity of inductively coupled plasma mass spectrometry (ICP-MS) allows identification and quantification of Np(IV) and Np(V) at the trace levels expected in the far field of a nuclear waste repository. Limits of detection of 1 × 10^-9 and 5 × 10^-10 mol L^-1 for Np(IV) and Np(V), respectively, were achieved, with a linear range from 10^-9 to 10^-6 mol L^-1. The method was applied to study the redox speciation of the Np remaining in solution after interaction of 5 × 10^-7 mol L^-1 Np(V) with Opalinus Clay. Under mildly oxidizing conditions, a Np sorption of 31% was found, with all the Np remaining in solution being Np(V). A second sorption experiment performed in the presence of Fe²⁺ led to complete sorption of the Np onto the clay. After desorption with HClO₄, a mixture of Np(IV) and Np(V) was found in solution by CE–ICP–MS, indicating that some of the sorbed Np had been reduced to Np(IV) by Fe²⁺.

Keywords: Capillary electrophoresis; Inductively coupled plasma mass spectrometry; Neptunium; Actinides; Speciation; “Hyphenated” techniques; Mira Mist CE nebulizer; Opalinus Clay; Sorption

Speciation of Np(V) uptake by Opalinus Clay using synchrotron microbeam techniques by Daniel R. Fröhlich; Samer Amayri; Jakob Drebert; Daniel Grolimund; Joachim Huth; Ugras Kaplan; Joachim Krause; Tobias Reich (pp. 2151-2162).

Synchrotron-based X-ray absorption spectroscopy has been used to determine the chemical speciation of Np sorbed on Opalinus Clay (OPA, Mont Terri, Switzerland), a natural argillaceous rock revealing a micro-scale heterogeneity. Different sorption and diffusion samples with Np(V) were prepared for spatially resolved molecular-level investigations. Thin sections of OPA contacted with Np(V) solution under aerobic and anaerobic conditions as well as a diffusion sample were analysed spatially resolved. Micro-X-ray fluorescence (μ-XRF) mapping has been used to determine the elemental distributions of Np, Fe and Ca. Regions of high Np concentration were subsequently investigated by micro-X-ray absorption fine structure spectroscopy to determine the oxidation state of Np. Further, micro-X-ray diffraction (μ-XRD) was employed to gain knowledge about reactive crystalline mineral phases in the vicinity of Np enrichments. One thin section was also analysed by electron microprobe to determine the elemental distributions of the lighter elements (especially Si and Al), which represent the main elements of OPA. The results show that in most samples, Np spots with considerable amounts of Np(IV) could be found even when the experiments were carried out in air. In some cases, almost pure Np(IV) L_III-edge X-ray absorption near-edge structure spectra were recorded. In the case of the anaerobic sample, the μ-XRF mapping showed a clear correlation between Np and Fe, indicating that the reduction of Np(V) is caused by an iron(II)-containing mineral which could be identified by μ-XRD as pyrite. These spatially resolved investigations were complemented by extended X-ray absorption fine structure measurements of powder samples from batch experiments under aerobic and anaerobic conditions to determine the structural parameters of the near-neighbour environment of sorbed Np.

Keywords: Neptunium; Opalinus Clay; μ-XAFS; μ-XRD; μ-XRF; EXAFS; Sorption; Speciation

Detection of plutonium isotopes at lowest quantities using in-source resonance ionization mass spectrometry by S. Raeder; A. Hakimi; N. Stöbener; N. Trautmann; K. Wendt (pp. 2163-2172).

The in-source resonance ionization mass spectrometry technique was applied for quantification of ultratrace amounts of plutonium isotopes as a proof of principle study. In addition to an overall detection limit of 10⁴ to 10⁵ atoms, this method enables the unambiguous identification and individual quantification of the plutonium isotopes ²³⁸Pu and ²⁴¹Pu which are of relevance for dating of radiogenic samples. Due to the element-selective ionization process, these isotopes can be measured even under a high surplus of isobaric contaminations from ²³⁸U or ²⁴¹Am, which considerably simplifies chemical preparation. The technique was developed, tested, and characterized on a variety of synthetic and calibration samples and is presently applied to analyze environmental samples.

Keywords: Resonance ionization mass spectrometry; In-source RIMS; Plutonium; Isotope ratios

Progress of ultra trace determination of technetium using laser resonance ionization mass spectrometry by K. Wendt; C. Geppert; C. Mattolat; G. Passler; S. Raeder; F. Schwellnus; K. Wies; N. Trautmann (pp. 2173-2176).

Laser resonance ionization mass spectrometry (RIMS) represents one of the most sensitive and selective techniques for ultra trace determination of long-lived radioisotopes. The isotope ^99gTc constitutes a specific candidate of high relevance concerning its environmental behavior as well as fundamental research applications. Based on the recent precision determination of the ionization potential of technetium by laser resonance ionization, refined resonant optical excitation pathways have been derived for analytical determination of ultra trace amounts of ^99gTc by laser mass spectrometric approaches. The state of the art and the specifications of RIMS-based ultra trace determination for ^99gTc, leading to a level of detection of ε ≈ 3 × 10 −⁴ atoms (3 μBq), are reported. Figure Detailed view of one of the Ti:sa lasers used for laser based ultra trace determination of technetium

Keywords: Resonance ionization spectroscopy; Mass spectrometry; RIMS; Technetium; Ultra trace detection

Generation of standard gas mixtures of halogenated, aliphatic, and aromatic compounds and prediction of the individual output rates based on molecular formula and boiling point by Ute R. Thorenz; Michael Kundel; Lars Müller; Thorsten Hoffmann (pp. 2177-2183).

In this work, we describe a simple diffusion capillary device for the generation of various organic test gases. Using a set of basic equations the output rate of the test gas devices can easily be predicted only based on the molecular formula and the boiling point of the compounds of interest. Since these parameters are easily accessible for a large number of potential analytes, even for those compounds which are typically not listed in physico-chemical handbooks or internet databases, the adjustment of the test gas source to the concentration range required for the individual analytical application is straightforward. The agreement of the predicted and measured values is shown to be valid for different groups of chemicals, such as halocarbons, alkanes, alkenes, and aromatic compounds and for different dimensions of the diffusion capillaries. The limits of the predictability of the output rates are explored and observed to result in an underprediction of the output rates when very thin capillaries are used. It is demonstrated that pressure variations are responsible for the observed deviation of the output rates. To overcome the influence of pressure variations and at the same time to establish a suitable test gas source for highly volatile compounds, also the usability of permeation sources is explored, for example for the generation of molecular bromine test gases.

Keywords: Test gas generation; Diffusive gas standard; Permeation gas standard; Theoretical calculation of output rates

HPLC-HG-ICP-MS: a sensitive and selective method for inorganic arsenic in seafood by Ásta H. Pétursdóttir; Helga Gunnlaugsdóttir; Hrönn Jörundsdóttir; Adrien Mestrot; Eva M. Krupp; Jörg Feldmann (pp. 2185-2191).

The addition of an online post-column hydride generation (HG) step to the commonly used high-performance liquid chromatography inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) setup for arsenic speciation proved to significantly improve the detection limits for the determination of inorganic arsenic (iAs) as arsenate in seafood samples, where the limit of detection and limit of quantification were found to be 0.0004 and 0.0014 mg kg⁻¹, respectively with HG. HG as an additional step further added to the selectivity of the determination of the iAs species and increased the detection and quantification of low levels of iAs (<0.002 mg kg⁻¹) in samples with complicated matrices.

Keywords: Speciation; Analytical chemistry; Seafood; Arsenic; Inorganic; CRM; Feed; Fish meal

Determination of perfluoroalkyl carboxylic, sulfonic, and phosphonic acids in food by Shahid Ullah; Tomas Alsberg; Robin Vestergren; Urs Berger (pp. 2193-2201).

A sensitive and accurate method was developed and validated for simultaneous analysis of perfluoroalkyl carboxylic acids, sulfonic acids, and phosphonic acids (PFPAs) at low picograms per gram concentrations in a variety of food matrices. The method employed extraction with acetonitrile/water and cleanup on a mixed-mode co-polymeric sorbent (C8 + quaternary amine) using solid-phase extraction. High-performance liquid chromatographic separation was achieved on a C18 column using a mobile phase gradient containing 5 mM 1-methyl piperidine for optimal chromatographic resolution of PFPAs. A quadrupole time-of-flight high-resolution mass spectrometer operating in negative ion mode was used as detector. Method detection limits were in the range of 0.002 to 0.02 ng g⁻¹ for all analytes. Sample preparation (extraction and cleanup) recoveries at a spiking level of 0.1 ng g⁻¹ to a baby food composite were in the range of 59 to 98 %. A strong matrix effect was observed in the analysis of PFPAs in food extracts, which was tentatively assigned to sorption of PFPAs to the injection vial in the solvent-based calibration standard. The method was successfully applied to a range of different food matrices including duplicate diet samples, vegetables, meat, and fish samples. Figure Extracted high-resolution mass chromatograms of a PFPAs spiked at 0.06 ng g ^–1 to baby food, b PFSAs spiked at 0.02 ng g ^–1 to baby food, and c PFCAs spiked at 0.02 ng g –1 to baby food

Keywords: PFCAs; PFSAs; PFPAs; Food samples; HPLC; HRMS

Isolation and characterization of degradation products of moxidectin using LC, LTQ FT-MS, H/D exchange and NMR by Atul Awasthi; Majid Razzak; Raida Al-Kassas; David R. Greenwood; Joanne Harvey; Sanjay Garg (pp. 2203-2222).

This study aimed to evaluate the degradation profile and pathways, and identify unknown impurities of moxidectin under stress conditions. During the experiments, moxidectin samples were stressed using acid, alkali, heat and oxidation, and chromatographic profiles were compared with known impurities given in European Pharmacopeia (EP) monograph. Moxidectin has shown good stability under heat, while reaction with alkali produced 2-epi and ∆2,3 isomers (impurities D and E in EP) by characteristic reactions of the oxahydrindene (hexahydrobenzofuran) portion of the macrocyclic lactone. Two new, previously unreported, unknown degradation products, i.e. impurity 1 and impurity 2, detected after acid hydrolysis of moxidectin (impurity 2 was also observed to a lesser extent after oxidation), were isolated from sample matrices and identified using liquid chromatography, NMR, high-resolution FT-ICR MS, and hydrogen/deuterium exchange studies. FTMS analysis showed accurate mass of molecular ion peaks for moxidectin at m/z 640.38412, impurity 1 at m/z 656.37952 and impurity 2 at m/z 611.35684, giving rise to daughter ions traceable up to the seventh levels of MSⁿ experiments and supporting the proposed structures. Both unknown impurities along with moxidectin were fully characterized by ¹H, ¹³C, 1D HMBC and 2D (NOESY, COSY and HSQC) NMR experiments. The interpretation of experimental data positively identified impurity 1 as 3,4-epoxy-moxidectin and impurity 2 as 23-keto-nemadectin. The identification of new impurities and correlation of their chromatographic profiles with the EP method is very useful to establish the stability profile of moxidectin and its preparations, as well as add value to the forthcoming moxidectin finished product European Pharmacopeia monographs. Figure Acid catalyzed degradation of moxidectin into 23-keto-moxidectin and 3,4-epoxy-moxidectin

Keywords: Moxidectin degradation product; High-resolution FT-MS and MSⁿ ; H/D exchange; NMR; Stress study; Macrocyclic lactone

Properties of coatings on RFID p-Chips that support plasmonic fluorescence enhancement in bioassays by Ryan Rich; Ji Li; Rafal Fudala; Zygmunt Gryczynski; Ignacy Gryczynski; Wlodek Mandecki (pp. 2223-2231).

Microtransponders (RFID p-Chips) derivatized with silver island film (SIF) have previously seen success as a platform for the quantification of low-abundance biomolecules in nucleic acid based assays and immunoassays. In this study, we further characterized the morphology of the SIF as well as the polymer matrix enveloping it by scanning electron microscopy (SEM). The polymer was a two-layer silane-based matrix engulfing the p-Chip and SIF. Through a series of SEM and confocal fluorescence microscopy experiments, we found the depth of the polymer matrix to be 1–2 μm. The radiative effects of the SIF/polymer layer were assessed by fluorescence lifetime imaging (FLIM) of p-Chips coated with the polymer to which a fluorophore (Alexa Fluor 555) was conjugated. FLIM images showed an 8.7-fold increase in fluorescence intensity and an increased rate of radiative decay, the latter of which is associated with improved photostability and both of which are linked to plasmonic enhancement by the SIF. Plasmonic enhancement was found to extend uniformly across the p-Chip and, interestingly, to a depth of about 1.2 μm. The substantial depth of enhancement suggests that the SIF/polymer layer constitutes a three-dimensional matrix that is accessible to solvent and small molecules such as fluorescent dyes. Finally, we confirmed that no surface-enhanced Raman scattering is seen from the SIF/polymer combination. The analysis provides a possible mechanism by which the SIF/polymer-coated p-Chips allow a highly sensitive immunoassay and, as a result, leads to an improved bioassay platform.

Keywords: ELISA; Multiplex; SIF; Surface plasmon resonance; FLIM; RFID

A DNA-conjugated magnetic nanoparticle assay for assessing genotoxicity by Colin D. Medley; Joshua E. Smith; Larry S. Wigman; Nik P. Chetwyn (pp. 2233-2239).

The genotoxicity of a molecule refers to its ability to interact with DNA in a way that inhibits normal DNA replication and transcription possibly leading to mutagenesis or carcinogenesis. Assessing the genotoxicity of a compound is critical in the development of pharmaceuticals and other products designed for human consumption or use. Typically genotoxicity is established using expensive and time consuming methods using animals or bacteria like the Ames test, mouse lymphoma assay, or mouse and rat carcinogenicity tests. We have developed a magnetic nanoparticle-based assay that uses conjugated double-stranded DNA to serve as a substrate for interaction with genotoxic molecules. After application of a magnetic field, the genotoxic molecules are extracted with the DNA-conjugated magnetic nanoparticles. The genotoxic molecules can then be released and detected. To evaluate the potential of this assay, we have screened several genotoxic and non-genotoxic compounds and have demonstrated the ability to extract a genotoxic compound in the presence of a non-genotoxic molecule. The assay demonstrates suitable analytical performance and the ability to differentiate between genotoxic and non-genotoxic molecules providing a rapid and inexpensive alternative to more traditional methods of evaluating genotoxicity.

Keywords: Nanoparticles; Nanotechnology; Nucleic acids (DNA | RNA); Pharmaceuticals

Differential immuno-kinetic assays of allergen-specific binding for peanut allergy serum analysis by Rouslan V. Olkhov; Edward R. Kaminski; Andrew M. Shaw (pp. 2241-2247).

A label-free nanoparticle array platform has been used to detect total peanut allergen-specific binding from whole serum of patients suffering from peanut allergy. The serum from 10 patients was screened against a four-allergen panel of cat and dog dander, dust mite and peanut allergen protein Ara h1. The IgE and IgG contributions to the total specific-binding protein load to Ara h1 were identified using two secondary IgG- and IgE-specific antibodies and were found to contribute less than 50 % of the total specific protein load. The total mass of IgE, IgE and the unresolved specific-binding protein ΔsBP for Ara h1 provides a new serum profile for high-RAST-grade patients 5 and 6 with the IgG/IgE ratio of 4 ± 2 and ΔsBP/IgE ratio of 17 ± 11, neither of which is protective for the small patient cohort. Figure Binding of the specefic IgE/IgG and unresolved material X to the sensor surface functionalized with allergen

Keywords: Ara h1; Array; Biosensor; IgE; IgG; Peanut allergy

Multireaction monitoring of 12 peptides for lowered immunity screening by Min-Jung Kang; Hyojeong Han; Oh-Seung Kwon; Hyun Ok Kim; Byung-Hwa Jung (pp. 2249-2258).

A multireaction monitoring method using high-performance liquid chromatography–tandem mass spectrometry was developed for 12 target peptides for determination of endogenous peptide concentrations in human serum. Chromatographic separation conditions were optimized and recoveries for liquid–liquid extraction, solid-phase extraction (SPE), and ultrafiltration of endogenous peptides from human serum were compared, and the SPE method was selected for 12 targeted peptide extractions. The optimized SPE method gave recoveries higher than 60 % for all targeted peptides. The limit of detection was 10 ng/ml for most peptides, except for N-formyl-methionyl-leucyl-phenylalanine (NFMLP) and adrenocorticotropic hormone (ACTH) (18-39). The limit of detection for these two peptides was 1 ng/ml. The real serum samples of 25 elderly and 23 young people were analyzed using the optimized extraction and analysis method. Half of the 12 peptides were below the limit of quantification, and B-type natriuretic peptide, cholecystokinin, ACTH(7–38), substance P, NFMLP, and valyl-glutamyl-prolyl-isoleucyl-prolyl-tyrosine were quantified in the concentration range from 0.1 to 50 ng/ml. The concentration of ACTH(7–38) was significantly higher in elderly people and that of NFMLP was significantly lower in elderly people compared with young people (p < 0.0001). This result implies that there be a possible relationships between ACTH, NFMLP and lowered immunity.

Keywords: Twelve endogenous peptides; Aging; Quantitative peptide profiling; High-performance liquid chromatography–tandem mass spectrometry; Serum; Biomarker discovery

Development, validation, and application of a liquid chromatography–tandem mass spectrometry method for the determination of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human hair by Li Yao; Jun Yang; Ya-feng Guan; Bai-zhan Liu; Sai-jing Zheng; Wei-miao Wang; Xiao-lan Zhu; Zhi-dan Zhang (pp. 2259-2266).

The tobacco-specific nitrosamine metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a valuable biomarker for human exposure to the carcinogenic nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in tobacco and tobacco smoke. In this work, an efficient and sensitive method for the analysis of NNAL in human hair was developed and validated. The hair sample was extracted by NaOH solution digestion, purified by C₁₈ solid-phase extraction (SPE) and molecularly imprinted solid-phase extraction, further enriched by reverse-phase ultrasound-assisted dispersive liquid–liquid microextraction (USA-DLLME) into 1.0 % aqueous formic acid, and finally analyzed by liquid chromatography–electrospray ionization tandem mass spectrometry. Good linearity was obtained in the range of 0.24–10.0 pg/mg hair with a correlation coefficient of 0.9982, when 150 mg hair was analyzed. The limit of detection and lower limit of quantification were 0.08 and 0.24 pg/mg hair, respectively. Accuracies determined from hair samples spiked with three different levels of NNAL ranged between 87.3 and 107.7 %. Intra- and inter-day relative standard deviations varied from 4.1 to 8.5 % and from 6.9 to 11.3 %, respectively. Under the optimized conditions, an enrichment factor of 20 was obtained. Finally, the developed method was applied for the analysis of NNAL in smokers' hair. The proposed sample preparation procedure combining selectivity of two-step SPE and enrichment of DLLME significantly improves the purification and enrichment of the analyte and should be useful to analyze NNAL in hair samples for cancer risk evaluation and cancer prevention in relation to exposure to the tobacco-specific carcinogen NNK. Figure An efficient analytical method based on LC-MS/MS detection has been developed and applied for analyzing the tobacco-specific carcinogen NNK metabolite NNAL in smokers’ hair.

Keywords: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol; Molecularly imprinted polymers; Solid-phase extraction; Dispersive liquid–liquid microextraction; Liquid chromatography tandem mass spectrometry; Human hair

Bead affinity chromatography in a temperature-controllable microsystem for biomarker detection by Yul Koh; Bo-Rahm Lee; Hyo-Jin Yoon; Yun-Ho Jang; Yoon-Sik Lee; Yong-Kweon Kim; Byung-Gee Kim (pp. 2267-2275).

This paper describes a temperature-controllable bead affinity chromatography (BAC) in a microsystem for biomarker detection, and preparing samples for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis. Cancer marker proteins were captured in the microsystem by BAC with RNA aptamer-immobilized microbeads. The captured proteins were then denatured and released from the microbeads by controlling temperature. The microsystem consists of a microreactor for trapping microbeads and a temperature control unit for thermal treatment of the trapped beads. We used polymethylsilxoane or single crystalline silicon in fabricating two different types of reaction chamber to compare the differences in performance originated from the materials. Carcinoembryonic antigen was concentrated and purified from human serum using the microsystem and detected by MALDI-TOF MS to demonstrate the usefulness of the microsystem. The microsystem simplifies a sample preparation process required for protein analysis and cancer biomarker detection, which will accelerate the process of cancer research.

Keywords: Temperature-controllable microfluidics; Aptamer-immobilized bead affinity chromatography; MALDI-TOF-MS; Carcinoembryonic antigen (CEA)

Sensitive, robust and automated protein analysis of cell differentiation and of primary human blood cells by intact cell MALDI mass spectrometry biotyping by Bogdan Munteanu; Carolina von Reitzenstein; Gertrud Maria Hänsch; Björn Meyer; Carsten Hopf (pp. 2277-2286).

Intact cell mass spectrometry biotyping, a collection of methods for classification of cells based on mass spectrometric fingerprints, is an established method in clinical and environmental microbiology. It has recently also been applied to the investigation of mammalian cells including primary blood cells and cultured cells. However, few automated procedures suitable for higher throughput and little analytical standardization of mammalian biotyping approaches have been reported so far. Here, we present a novel automated method that robustly classifies as few as 250 cells per spot. Automatically acquired cell fingerprints from cultured and primary cells show high technical (R > 0.95) and biological reproducibility (R = 0.83–0.96), with a median peak variance below 12 %. Ion suppression is shown to be a major concern at higher cell numbers and needs to be carefully monitored. We demonstrate that intact cell mass spectrometric signatures of different cell lines start to resemble each other at higher trifluoroacetic acid (TFA) concentrations and that therefore low concentrations of TFA in the matrix solution are preferred. We show that in vitro differentiation of HL-60 cells into a neutrophil-like phenotype can be rapidly and robustly monitored. We utilize the method for global analysis of person-to-person differences in mass spectral signatures of intact polymorphonuclear neutrophils and monocytes obtained from healthy volunteers. Our data suggest that automated MALDI mass spectrometry cell biotyping could be a useful complementary approach in clinical cell analysis. Figure Sensitive, robust and automated MALDI mass spectrometry biotyping enables analysis of cell differentiation fingerprints

Keywords: Intact cell mass spectrometry; MALDI; Mammalian cell biotyping; Cell differentiation; Primary blood cells

Simultaneous determination of cyanide and thiocyanate in plasma by chemical ionization gas chromatography mass-spectrometry (CI-GC-MS) by Raj K. Bhandari; Robert P. Oda; Stephanie L. Youso; Ilona Petrikovics; Vikhyat S. Bebarta; Gary A. Rockwood; Brian A. Logue (pp. 2287-2294).

An analytical method utilizing chemical ionization gas chromatography-mass spectrometry was developed for the simultaneous determination of cyanide and thiocyanate in plasma. Sample preparation for this analysis required essentially one-step by combining the reaction of cyanide and thiocyanate with pentafluorobenzyl bromide and simultaneous extraction of the product into ethyl acetate facilitated by a phase-transfer catalyst, tetrabutylammonium sulfate. The limits of detection for cyanide and thiocyanate were 1 μM and 50 nM, respectively. The linear dynamic range was from 10 μM to 20 mM for cyanide and from 500 nM to 200 μM for thiocyanate with correlation coefficients higher than 0.999 for both cyanide and thiocyanate. The precision, as measured by %RSD, was below 9 %, and the accuracy was within 15 % of the nominal concentration for all quality control standards analyzed. The gross recoveries of cyanide and thiocyanate from plasma were over 90 %. Using this method, the toxicokinetic behavior of cyanide and thiocyanate in swine plasma was assessed following cyanide exposure.

Keywords: Cyanide; Thiocyanate; Method development; Chemical-ionization gas-chromatography mass-spectrometry

Quantitation of intracellular purine intermediates in different Corynebacteria using electrospray LC-MS/MS by Susanne Peifer; Konstantin Schneider; Gudrun Nürenberg; Dietrich A. Volmer; Elmar Heinzle (pp. 2295-2305).

Intermediates of the purine biosynthesis pathway play key roles in cellular metabolism including nucleic acid synthesis and signal mediation. In addition, they are also of major interest to the biotechnological industry as several intermediates either possess flavor-enhancing characteristics or are applied in medical therapy. In this study, we have developed an analytical method for quantitation of 12 intermediates from the purine biosynthesis pathway including important nucleotides and their corresponding nucleosides and nucleobases. The approach comprised a single-step acidic extraction/quenching procedure, followed by quantitative electrospray LC-MS/MS analysis. The assay was validated in terms of accuracy, precision, reproducibility, and applicability for complex biological matrices. The method was subsequently applied for determination of free intracellular pool sizes of purine biosynthetic pathway intermediates in the two Gram-positive bacteria Corynebacterium glutamicum and Corynebacterium ammoniagenes. Importantly, no ion pair reagents were applied in this approach as usually required for liquid chromatography analysis of large classes of diverse metabolites.

Keywords: Biological samples; HPLC; Mass spectrometry; Nucleic acid sampling; Quantitative extraction; Bioanalytical methods

Spectroscopic analysis of bacterial biological warfare simulants and the effects of environmental conditioning on a bacterial spectrum by Alastair J. S. McIntosh; Stephen J. Barrington; Hilary Bird; Daniel Hurst; Phillippa Spencer; Suzanne H. Pelfrey; Matthew J. Baker (pp. 2307-2315).

The ability to distinguish bacteria from mixed samples is of great interest, especially in the medical and defence arenas. This paper reports a step towards the aim of differentiating pathogenic endospores in situ, to aid any required response for hazard management using infrared spectroscopy combined with multivariate analysis. We describe a proof-of-principle study aimed at discriminating biological warfare simulants from common environmental bacteria. We also report an evaluation of multiple pre-processing techniques and subsequent differences in cross-validation of two pattern recognition models (Support Vector Machines and Principal Component–Linear Discriminant Analysis) for a six-class classification (bacterial classification). These classifications were possible with an average sensitivity of 88.0 and 86.9 %, and an average specificity of 97.6 and 97.5 % for the SVM and the PC-LDA models, respectively. Most spectroscopic models are built upon spectra from bacteria that have been specifically prepared for analysis by a particular method; this paper will comment upon the differences in the bacterial spectrum that occur between specific preparations when the bacteria have spent 30 days in the simulated weather conditions of a hot dry climate. Figure 3D-PC-LDA score plot (left), for the six class bacterial identification, with the loading plot (right) for each of the three discriminant functions used

Keywords: Infrared spectroscopy; Bacteria; Biological warfare; Surface deposited Bacillus ; Pre-processing

Evaluating the validity of spectral calibration models for quantitative analysis following signal preprocessing by Da Chen; Edward Grant (pp. 2317-2327).

When paired with high-powered chemometric analysis, spectrometric methods offer great promise for the high-throughput analysis of complex systems. Effective classification or quantification often relies on signal preprocessing to reduce spectral interference and optimize the apparent performance of a calibration model. However, less frequently addressed by systematic research is the affect of preprocessing on the statistical accuracy of a calibration result. The present work demonstrates the effectiveness of two criteria for validating the performance of signal preprocessing in multivariate models in the important dimensions of bias and precision. To assess the extent of bias, we explore the applicability of the elliptic joint confidence region (EJCR) test and devise a new means to evaluate precision by a bias-corrected root mean square error of prediction. We show how these criteria can effectively gauge the success of signal pretreatments in suppressing spectral interference while providing a straightforward means to determine the optimal level of model complexity. This methodology offers a graphical diagnostic by which to visualize the consequences of pretreatment on complex multivariate models, enabling optimization with greater confidence. To demonstrate the application of the EJCR criterion in this context, we evaluate the validity of representative calibration models using standard pretreatment strategies on three spectral data sets. The results indicate that the proposed methodology facilitates the reliable optimization of a well-validated calibration model, thus improving the capability of spectrophotometric analysis. Graphical Abstract Elliptic Joint Condence Region plots obtained following methods for preprocessing spectra of lactic acid in water. Models derived from raw spectra (■), and spectra following geometric pretreatment methods: Multiplicative Signal Correction (o), Savitzky-Golay-1D (▲), Savitzky-Golay-2D (◊), Discrete Wavelet Transform (*) and Continuous Wavelet Transform(+).

Keywords: Raman spectroscopy; Spectrochemical analysis; Feature selection; Precision; Bias

Characterization and deposition of various light-harvesting antenna complexes by electrospray atomization by Vivek B. Shah; Gregory S. Orf; Sean Reisch; Lucas B. Harrington; Mindy Prado; Robert E. Blankenship; Pratim Biswas (pp. 2329-2338).

Photosynthetic organisms have light-harvesting complexes that absorb and transfer energy efficiently to reaction centers. Light-harvesting complexes (LHCs) have received increased attention in order to understand the natural photosynthetic process and also to utilize their unique properties in fabricating efficient artificial and bio-hybrid devices to capture solar energy. In this work, LHCs with different architectures, sizes, and absorption spectra, such as chlorosomes, Fenna–Matthews–Olson (FMO) protein, LH2 complex, and phycobilisome have been characterized by an electrospray-scanning mobility particle-sizer system (ES-SMPS). The size measured by ES-SMPS for FMO, chlorosomes, LH2, and phycobilisome were 6.4, 23.3, 9.5, and 33.4 nm, respectively. These size measurements were compared with values measured by dynamic light scattering and those reported in the literature. These complexes were deposited onto a transparent substrate by electrospray deposition. Absorption and fluorescence spectra of the deposited LHCs were measured. It was observed that the LHCs have light absorption and fluorescence spectra similar to that in solution, demonstrating the viability of the process.

Keywords: Electrospray-scanning mobility particle sizer (ES-SMPS); Electrospray deposition; Light-harvesting complexes (LHCs)

Unraveling the role of membrane proteins Notch, Pvr, and EGFR in altering integrin diffusion and clustering by Neha Arora; Dipak Mainali; Emily A. Smith (pp. 2339-2348).

The role of three membrane proteins in altering the diffusion and clustering of integrin receptors has been measured. Integrins are membrane proteins responsible for integrating intracellular and extracellular signaling events and anchoring cells to the extracellular matrix. The methodology used to elucidate the role of other membrane proteins in altering integrin diffusion and clustering combines fluorescence microscopy with RNA interference (RNAi), which is a technique to reduce the expression of a target protein. The three RNAi-targeted membrane proteins were epidermal growth factor receptor (EGFR), platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr), and Notch. Real-time polymerase chain reaction or quantitative immunocytochemistry was used to measure a reduction in mRNA or protein concentration after RNAi treatment, respectively. Fluorescence recovery after photobleaching showed that reducing the concentration of EGFR or Notch results in less constrained integrin diffusion and, in the case of Notch RNAi, 4 % more mobile integrins. Fluorescence resonance energy transfer measurements performed before and after RNAi treatments indicate that clustering decreases for wild-type integrin, but increases for a high-ligand-affinity integrin mutant after reducing the expression of EGFR, Pvr, or Notch. A model to explain the measured changes after reducing the expression of these three membrane proteins involving cholesterol-enriched nanodomains is proposed. Figure A combination of fluorescence microscopy and RNAi interference were used to measure how selectively reducing the expression of one membrane protein affects clustering and diffusion of another membrane protein

Keywords: RNA interference; Fluorescence recovery after photobleaching (FRAP); Fluorescence resonance energy transfer (FRET); Membrane nanodomains; Lipid diffusion; Fluorescence microscopy

SPE–NMR metabolite sub-profiling of urine by Doris M. Jacobs; Laura Spiesser; Maxime Garnier; Niels de Roo; Ferdi van Dorsten; Boudewijn Hollebrands; Ewoud van Velzen; Richard Draijer; John van Duynhoven (pp. 2349-2361).

NMR-based metabolite profiling of urine is a fast and reproducible method for detection of numerous metabolites with diverse chemical properties. However, signal overlap in the ¹H NMR profiles of human urine may hamper quantification and identification of metabolites. Therefore, a new method has been developed using automated solid-phase extraction (SPE) combined with NMR metabolite profiling. SPE–NMR of urine resulted in three fractions with complementary and reproducible sub-profiles. The sub-profile from the wash fraction (100 % water) contained polar metabolites; that from the first eluted fraction (10 % methanol–90 % water) semi-polar metabolites; and that from the second eluted fraction (100 % methanol) aromatic metabolites. The method was validated by analysis of urine samples collected from a crossover human nutritional intervention trial in which healthy volunteers consumed capsules containing a polyphenol-rich mixture of red wine and grape juice extract (WGM), the same polyphenol mixture dissolved in a soy drink (WGM_Soy), or a placebo (PLA), over a period of five days. Consumption of WGM clearly increased urinary excretion of 4-hydroxyhippuric acid, hippuric acid, 3-hydroxyphenylacetic acid, homovanillic acid, and 3-(3-hydroxyphenyl)-3-hydroxypropionic acid. However, there was no difference between the excreted amounts of these metabolites after consumption of WGM or WGM_Soy, indicating that the soy drink is a suitable carrier for WGM polyphenols. Interestingly, WGM_Soy induced a significant increase in excretion of cis-aconitate compared with WGM and PLA, suggesting a higher demand on the tricarboxylic acid cycle. In conclusion, SPE–NMR metabolite sub-profiling is a reliable and improved method for quantification and identification of metabolites in urine to discover dietary effects and markers of phytochemical exposure. Figure SPE-NMR sub-profiles of urine compared to reconstructed (blue) and non-fractionated (reference) profile

Keywords: Metabolomics; NMR spectroscopy; Metabolite profiling; Urine; Polyphenol; Wine; Soy

Chiral separation and determination of excitatory amino acids in brain samples by CE-LIF using dual cyclodextrin system by Zsolt Wagner; Tamás Tábi; Tamás Jakó; Gergely Zachar; András Csillag; Éva Szökő (pp. 2363-2368).

Chiral capillary electrophoresis method has been developed to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of d-Asp in the central nervous system. To achieve appropriate detection sensitivity fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and laser-induced fluorescence detection was applied. Although, simultaneous baseline separation of the two enantiomer pairs could be achieved by using 3 mM 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-cyclodextrin (HPA-β-CD), further improvement of the chemical selectivity was required because of the high excess of l-enantiomers in real samples to be analyzed. The system selectivity was fine-tuned by combination of 8 mM heptakis(2,6-di-O-methyl)-β-cyclodextrin and 5 mM HPA-β-CD in order to increase the resolution between aspartate and glutamate enantiomers. The method was validated for biological application. The limits of detection for d-Asp and d-Glu were 17 and 9 nM, respectively, while the limit of quantification for both analytes was 50 nM. This is the lowest quantification limit reported so far for NBD-tagged d-Asp and d-Glu obtained by validated capillary electrophoresis laser-induced fluorescence method. The applicability of the method was demonstrated by analyzing brain samples of 1-day-old chickens. In all the studied brain areas, the d-enantiomer contributed 1–2 % of the total aspartate content, corresponding to 17–45 nmol/g wet tissue.

Keywords: Chiral capillary electrophoresis; Neurotransmitter amino acids; d-Aspartate; NBD-F; Dual cyclodextrin system

Real-time determination of the activity of ATPase by use of a water-soluble polythiophene by Umit Hakan Yildiz; Chia Wei Sheng; Diyar Mailepessov; Diana Chia Xueqi; Susana Geifman Shochat; Bo Liedberg (pp. 2369-2375).

This contribution introduces a fluorescence assay for real-time determination of the activity of p97/VCP, a 540-kDa homo-hexameric enzyme, belonging to the AAA-ATPase family. A fluorescent reporter “poly 1-(3-((4-methylthiophen-3-yl)oxy)propyl)quinuclidin-1-ium” (poly PTQ) is used to monitor the hydrolysis of ATP to ADP by p97/VCP. The proposed assay relies on the different strength of coordination of ATP and ADP to the polymer backbone. We used recovery of fluorescence intensity on addition of p97/VCP to a poly PTQ/ATP solution to determine the enzymatic activity. The kinetic data K _m and V _max were 0.30 mmol L⁻¹ ATP and 0.134 nmol ATP min⁻¹ μg⁻¹ enzyme, respectively. The specificity of the assay was investigated by using an unhydrolyzable ATP analogue and sensitivity against p97 mutagenesis was further examined by detection of the activity of wild type and truncated p97/VCP. Our study demonstrates that determination of the real-time activity of p97/VCP is possible, because of the superior sensitivity and very fast optical response of poly PTQ. Figure Illustration of fluorescence based detection mechanism for real-time determination of the activity of ATPase by water-soluble polythiophene

Keywords: Real-time activity determination; Fluorescence recovery; Cationic polythiophene

Use of crude extract of lentil plant (Lens culinaris Medikus) in peroxidase-based analyses: fast kinetic determination of hydrogen peroxide and sarcosine in urine by Patricia Pérez Galende; Teresa Manzano Muñoz; Manuel G. Roig; Cándido García de María (pp. 2377-2385).

Peroxidase-catalysed reactions are used in a wide variety of analytical applications, most of them based on the final quantification of hydrogen peroxide. Clinical tests for glucose, cholesterol, creatine, creatinine or uric acid in blood or urine and enzyme-linked immunosorbent assays for pesticides, hepatitis or acquired immune deficiency syndrome are good examples of such applications. The most widely used and commercially available peroxidase for biotechnological processes and analytical applications is horseradish peroxidase followed, although in much lower proportion, by soybean peroxidase. The high commercial interest in peroxidases has led to the search for new sources of these enzymes. This work describes the analytical use of lentil plant peroxidase (LPP), which is a new peroxidase extracted from lentil plants (Lens culinaris Medikus); an abundant post-harvest agricultural waste in the area of Castilla y León (Spain). A procedure for the quantification of hydrogen peroxide in urine is first proposed using crude extract of lentil plant instead of the purified enzyme. This procedure is then applied to the determination of sarcosine; a natural amino acid that has attracted considerable interest in clinical diagnostics since urinary sarcosine was proposed and later questioned as a biomarker for prostate cancer. Under the action of sarcosine oxidase, sarcosine is oxidized by molecular oxygen to give glycine, formaldehyde and hydrogen peroxide that is quantified according to the previously proposed procedure. The limit of detection for both hydrogen peroxide and sarcosine is around 5 × 10⁻⁷ M. In the determination of sarcosine, the high selectivity of the overall enzymatic reaction, the simple sample treatment and instrumentation, the high-sample throughput and the use of LPP in the plant extract instead of the purified enzyme provide a rapid and inexpensive procedure with characteristics very suitable for routine analysis in a clinical laboratory. Figure Lentil plant of ‘Rubia de la Armuña’ (Lens culinaris Medikus)/Determination of sarcosine

Keywords: Lentil plant peroxidase; Plant extract; Hydrogen peroxide; Sarcosine; Urine; Prostate cancer

Qualitative analysis of the fluorophosphonate-based chemical probes using the serine hydrolases from mouse liver and poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis by Yi-Long Huang; Tsai-Wen Chung; Chia-Mao Chang; Chih-Hau Chen; Chen-Chung Liao; Yeou-Guang Tsay; Gwo-Chyuan Shaw; Shwu-Huey Liaw; Chung-Ming Sun; Chao-Hsiung Lin (pp. 2387-2396).

The serine hydrolase family consists of more than 200 members and is one of the largest enzyme families in the human genome. Although up to 50 % of this family remains unannotated, there are increasing evidences that activities of certain serine hydrolases are associated with diseases like cancer neoplasia, invasiveness, etc. By now, several activity-based chemical probes have been developed and are applied to profile the global activity of serine hydrolases in diverse proteomes. In this study, two fluorophosphonate (FP)-based chemical probes were synthesized. Further examination of their abilities to label and pull down serine hydrolases was conducted. In addition, the poly-3-hydroxybutyrate depolymerase (PhaZ) from Bacillus thuringiensis was demonstrated as an appropriate standard serine hydrolase, which can be applied to measure the labeling ability and pull-down efficiency of FP-based probes. Furthermore, mass spectrometry (MS) was used to identify the serine residue that covalently bonded to the active probes. Finally, these FP-based probes were shown capable of establishing the serine hydrolase profiles in diverse mouse tissues; the serine hydrolases pulled down from mouse liver organ were further identified by MS. In summary, our study provides an adequate method to evaluate the reactivity of FP-based probes targeting serine hydrolases. Figure

Keywords: Activity-based probe; Serine hydrolase; PhaZ; Esterase; Fluorophosphonate

On-line immobilized acetylcholinesterase microreactor for screening of inhibitors from natural extracts by capillary electrophoresis by Wenao Min; Weiping Wang; Jianrong Chen; Aijun Wang; Zhide Hu (pp. 2397-2405).

In this study we developed a simple capillary electrophoresis (CE) method with an on-line acetylcholinesterase (AChE) microreactor at the inlet of capillary for inhibitor screening. The fused-silica capillary surface was modified with a polycationic polyethylenimine coating. Solutions of the enzyme and chitosan were then injected to immobilize the enzyme in approximately 2.9 cm of the capillary inlet (total length of capillary 60.2 cm) by electrostatic interaction and the film overlay technique. Separation of enzyme reaction product (thiocholine, ThCh) and unreacted substrate (acetylthiocholine, AThCh) was achieved within 3.0 min. The conditions affecting the efficiency of reaction of the enzyme were optimized by measuring the peak area of ThCh. Under the optimum conditions, using Huperzine-A as model inhibitor, K _i and IC ₅₀ were 0.551 μmol L⁻¹ and 1.52 μmol L⁻¹, respectively, for immobilized AChE. Finally, screening of a small compound library containing two known AChE inhibitors and 30 natural extracts was conducted, and species with inhibition activity were directly identified. Compared with previous publications on screening for AChE inhibitors in natural products based on CE methods, the method developed in this work has the advantages of lower cost per analysis, less leakage, and better bioaffinity for the immobilized enzyme because of the unique properties of sodium alginate and chitosan. Figure (a) Schematic representation of the immobilized enzymatic microreactor. (b) Typical electropherograms for AChE enzymatic hydrolysis reaction. Peaks: S, substrate (AThCh); P, product (ThCh). CE conditions: fused-silica capillary, dimensions, 75 μm × 60.2 cm (50 cm to detection window); running buffer, 20 mM Tris-HCl (pH 8.0); cartridge temperature, 37 ℃; sample injection, 0.5 psi, 10 s; incubation time, 2 min; separation potential, 25 kV; detection wavelength, 230 nm; concentrations of AChE and substrate: 0.37 mg mL⁻¹ (containing 20 mM MgSO₄ and 1.0 g L⁻¹ sodium alginate) and 10 mM, respectively.

Keywords: Acetylcholinesterase; Immobilization; Capillary electrophoresis; Inhibitor screening; Natural extracts

Separation mechanism of chiral impurities, ephedrine and pseudoephedrine, found in amphetamine-type substances using achiral modifiers in the gas phase by Howard K. Holness; Adeel Jamal; Alexander Mebel; José R. Almirall (pp. 2407-2416).

A new mechanism is proposed that describes the gas-phase separation of chiral molecules found in amphetamine-type substances (ATS) by the use of high-resolution ion mobility spectrometry (IMS). Straight-chain achiral alcohols of increasing carbon chain length, from methanol to n-octanol, are used as drift gas modifiers in IMS to highlight the mechanism proposed for gas-phase separations of these chiral molecules. The results suggest the possibility of using these achiral modifiers to separate the chiral molecules (R,S) and (S,R)-ephedrine and (S,S) and (R,R)-pseudoephedrine which contain an internal hydroxyl group at the first chiral center and an amino group at the other chiral center. Ionization was achieved with an electrospray source, the ions were introduced into an IMS with a resolving power of 80, and the resulting ion clusters were characterized with a coupled quadrupole mass spectrometer detector. A complementary computational study conducted at the density functional B3LYP/6-31g level of theory for the electronic structure of the analyte–modifier clusters was also performed, and showed either “bridged” or “independent” binding. The combined experimental and simulation data support the proposed mechanism for gas-phase chiral separations using achiral modifiers in the gas phase, thus enhancing the potential to conduct fast chiral separations with relative ease and efficiency.

Keywords: Chiral; Ion mobility spectrometry; Electrospray; Amphetamines; Mass spectrometry; Density function theory

Fragment screening of cyclin G-associated kinase by weak affinity chromatography by Elinor Meiby; Stefan Knapp; Jonathan M. Elkins; Sten Ohlson (pp. 2417-2425).

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K _D) = mM − μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target—cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K _D < 200 μM were detected, including a few highly efficient GAK binders (K _D of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

Keywords: Fragment screening; Fragment-based drug discovery; Cyclin G-associated kinase; Weak affinity chromatography

Simultaneous enantiomeric determination of MDMA and its phase I and phase II metabolites in urine by liquid chromatography–tandem mass spectrometry with chiral derivatization by Keiko Nakanishi; Munehiro Katagi; Kei Zaitsu; Noriaki Shima; Hiroe Kamata; Akihiro Miki; Hajime Kato; Ken-Ichi Harada; Hitoshi Tsuchihashi; Koichi Suzuki (pp. 2427-2435).

A liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) procedure was developed for the simultaneous determination of enantiomers of the prevalent designer drug 3,4-methylenedioxymethamphetamine (MDMA) and its phase I and phase II metabolites in urine with chiral derivatization. The analytes in urine were directly derivatized with chiral Marfey’s reagent, N ^α- (5-fluoro-2,4-dinitrophenyl)-d-leucinamide, without extraction. The diastereomers of the N ^α-(2,4-dinitrophenyl)-d-leucinamide derivatives generated were determined by LC-MS/MS. Satisfactory chromatographic separation was achieved for the enantiomers of MDMA and its metabolites 3,4-methylenedioxyamphetamine, 4-hydroxy-3-methoxymethamphetamine (HMMA), HMMA glucuronide, and HMMA sulfate on a semimicro octadecylsilane column using linear gradient elution. With use of multiple reaction monitoring mode, the limits of detection of these analytes ranged from 0.01 to 0.03 μg/mL. Linear calibration curves were obtained for all enantiomers from 0.1 to 20 μg/mL in urine. The method showed sufficient reproducibility and quantitative ability. This is the first report of a simple LC-MS/MS-based analytical procedure with direct chiral derivatization in aqueous media that allows simultaneous enantiomeric determination of drugs and their metabolites, including glucuronide and sulfate derivatives.

Keywords: MDMA; Enantiomer; Liquid chromatography–tandem mass spectrometry; Phase II metabolite; Marfey’s reagent

Structural stability–chromatographic retention relationship on exenatide diastereomer separation by Ching-Wei Tsai; Wei-Hung Kao; Li-Chiao Chang; Ruoh-Chyu Ruaan; Wen-Yih Chen (pp. 2437-2444).

In this study, the relationship of the structural stability of peptide diastereomers in elution solvents and their retention behaviors in reversed-phase chromatography (RPC) was examined to provide guidance on the solvent selection for a better separation of peptide diastereomers. We investigated the chromatographic retention behaviors of exenatide, a peptide drug for the treatment of type II diabetes mellitus and its three diastereomers using RPC and implicit molecular dynamics (MD) simulation analysis. Three diastereomers involved in the single serine residue mutation of d-form at the 11th, 32nd, and 39th residues were investigated in this study. Results show that the order of the solution structural stability of exenatide and its diastereomers is consistent with their retention order by 36 % acetonitrile/water elution. The sample loading solvent also affects the retention behaviors of exenatide peptide diastereomers in RPC column. Furthermore, a larger solution conformation energy difference of the critical pair of exenatide and its diastereomer (d-Ser39) at the elution solvent of 32 % tetrahydrofuran/water were obtained by MD simulation, and baseline separation was proved experimentally. In summary, we demonstrated that the solution structural stability–chromatographic retention relationship could be a powerful tool for elution solvent selection in peptide chromatographic purification, especially valuable for the separation of critical pair of diastereomers. Figure The structural stability and reversed-phase chromatography (RPC) retention relationship was investigated for a better chromatographic separation of peptides. Our results revealed that the rigid peptide with lower solution conformation energy exhibits a smaller retention factor in RPC column. Conversely, the flexible peptide with the higher solution conformation energy exhibits a larger retention factor. Based on this finding, we have examined that the baseline separation could be achieved by tuning the elution solvent composition to increase the structural stability difference between peptides. Consequently, the structural stability and RPC retention relationship could actually provide an important guidance on peptide separation.

Keywords: Exenatide; Diastereomer separation; Reversed-phase chromatography; Molecular dynamics simulation; Structural stability–chromatographic retention relationship

Dual-bioactivity-based liquid chromatography-coupled quadrupole time-of-flight mass spectrometry for NF-κB inhibitors and β₂AR agonists identification in Chinese Medicinal Preparation Qingfei Xiaoyan Wan by Binfeng Cheng; Yuanyuan Hou; Liqiang Wang; Linyi Dong; Jiamin Peng; Gang Bai (pp. 2445-2452).

Traditional Chinese medicine (TCM) preparations have been used as an effective multitarget strategy for the treatment of complex diseases; however, their bioactive constituents are undefined and difficult to identify. In this study, a simple and dual-target method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry combined with dual-bioactive (NF-κB and β₂-adrenergic receptor) luciferase reporter assay systems was developed for the rapid determination of various bioactive compounds of TCM preparations. Qingfei Xiaoyan Wan, a TCM preparation used for the clinical therapy of asthma, was analyzed with this method. Potential anti-inflammatory and spasmolytic constituents were screened using NF-κB and β₂-adrenergic receptor activity luciferase reporter assay systems and simultaneously identified according to the time-of-flight mass spectrometry data. One β₂-adrenergic receptor agonist (ephedrine) and four structural types of NF-κB inhibitors (arctigenin derivatives, cholic acid derivatives, chlorogenic acid, and sinapic acid) were characterized. Tracheloside was considered a new NF-κB inhibitor. Further cytokine and chemokine detection confirmed the anti-inflammatory effects of the potential NF-κB inhibitors. The integration of ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and dual-bioactive human cell functional evaluation systems proved to be a simple and effective strategy for the rapid screening of various bioactive compounds in TCM preparations used to treat complex diseases.

Keywords: Dual-bioactive target; Constituent identification; β₂ adrenergic receptor agonists; NF-κB inhibitors; UPLC/Q-TOF-MS; Qingfei Xiaoyan Wan

Determination of bisphenol A and bisphenol B in canned seafood combining QuEChERS extraction with dispersive liquid–liquid microextraction followed by gas chromatography–mass spectrometry by S. C. Cunha; C. Cunha; A. R. Ferreira; J. O. Fernandes (pp. 2453-2463).

A new simple and reliable method combining an acetonitrile partitioning extractive procedure followed by dispersive solid-phase cleanup (QuEChERS) with dispersive liquid–liquid microextraction (DLLME) and further gas chromatography mass spectrometry analysis was developed for the simultaneous determination of bisphenol A (BPA) and bisphenol B (BPB) in canned seafood samples. Besides the great enrichment factor provided, the final DLLME extractive step was designed in order to allow the simultaneous acetylation of the compounds required for their gas chromatographic analysis. Tetrachloroethylene was used as extractive solvent, while the acetonitrile extract obtained from QuEChERS was used as dispersive solvent, and anhydride acetic as derivatizing reagent. The main factors influencing QuEChERS and DLLME efficiency including nature of QuEChERS dispersive-SPE sorbents, amount of DLLME extractive and dispersive solvents and nature and amount of derivatizing reagent were evaluated. DLLME procedure provides an effective enrichment of the extract, allowing the required sensitivity even using a single quadropole MS as detector. The optimized method showed to be accurate (>68 % recovery), reproducible (<21 % relative standard deviation) and sensitive for the target analytes (method detection limits of 0.2 μg/kg for BPA and 0.4 μg/kg for BPB). The screening of several canned seafood samples commercialized in Portugal (total = 47) revealed the presence of BPA in more than 83 % of the samples with levels ranging from 1.0 to 99.9 μg/kg, while BPB was found in only one sample at a level of 21.8 μg/kg.

Keywords: DLLME (dispersive liquid–liquid microextraction); BPA (bisphenol A); BPB (bisphenol B); Canned food; Gas chromatography–mass spectrometry (GC–MS)

Development and independent laboratory validation of a simple method for the determination of paraquat and diquat in potato, cereals and pulses by Diana I. S. Kolberg; Dorothea Mack; Michelangelo Anastassiades; Michael T. Hetmanski; Richard J. Fussell; Thijs Meijer; Hans G. J. Mol (pp. 2465-2474).

A new sensitive, fast and robust method for the determination of paraquat and diquat residues in potatoes, cereals and pulses is presented. Different extraction conditions (solvent, time and temperature) have been evaluated using barley grain, potatoes and dry lentils containing incurred residues of diquat and paraquat. The finalised procedure involves extraction with a mixture of methanol/water/hydrochloric acid at 80 °C and analysis by liquid chromatography–tandem mass spectrometry. Diquat D4 and Paraquat D6 internal standards were added to the test portions prior to extraction. A small-scale inter-laboratory validation of the developed method for diquat and paraquat using potato and barley samples was conducted by three laboratories. The precision and accuracy of the method were determined from recovery experiments (five replicates) at 0.01 and 0.1 mg kg⁻¹. The recoveries obtained (n = 180) were in the range of 92–120 % with associated relative standard deviation (RSD) between 1.4–10 % for all compound/commodity/spiking concentration combinations.

Keywords: Diquat; Paraquat; Potato; Cereals; Lentils; LC-MS/MS

Is it possible to agree on a value for inorganic arsenic in food? The outcome of IMEP-112 by M. B. de la Calle; I. Baer; P. Robouch; F. Cordeiro; H. Emteborg; M. J. Baxter; N. Brereton; G. Raber; D. Velez; V. Devesa; R. Rubio; T. Llorente-Mirandes; A. Raab; J. Feldmann; J. J. Sloth; R. R. Rasmussen; M. D’Amato; F. Cubadda (pp. 2475-2488).

Two of the core tasks of the European Union Reference Laboratory for Heavy Metals in Feed and Food (EU-RL-HM) are to provide advice to the Directorate General for Health and Consumers (DG SANCO) on scientific matters and to organise proficiency tests among appointed National Reference Laboratories. This article presents the results of the 12th proficiency test organised by the EU-RL-HM (IMEP-112) that focused on the determination of total and inorganic arsenic in wheat, vegetable food and algae. The test items used in this exercise were: wheat sampled in a field with a high concentration of arsenic in the soil, spinach (SRM 1570a from NIST) and an algae candidate reference material. Participation in this exercise was open to laboratories from all around the world to be able to judge the state of the art of the determination of total and, more in particular, inorganic arsenic in several food commodities. Seventy-four laboratories from 31 countries registered to the exercise; 30 of them were European National Reference Laboratories. The assigned values for IMEP-112 were provided by a group of seven laboratories expert in the field of arsenic speciation analysis in food. Laboratory results were rated with z and ζ scores (zeta scores) in accordance with ISO 13528. Around 85 % of the participants performed satisfactorily for inorganic arsenic in vegetable food and 60 % did for inorganic arsenic in wheat, but only 20 % of the laboratories taking part in the exercise were able to report satisfactory results in the algae test material.

Keywords: Wheat; Vegetables; Algae; Inorganic arsenic

Degradation and epimerization of ergot alkaloids after baking and in vitro digestion by Stefan Merkel; Baha Dib; Ronald Maul; Robert Köppen; Matthias Koch; Irene Nehls (pp. 2489-2497).

The degradation and epimerization of ergot alkaloids (EAs) in rye flour were investigated after baking cookies and subsequently subjecting them to an in vitro digestion model. Different steps of digestion were analyzed using salivary, gastric, and duodenal juices. The degradation and bidirectional conversion of the toxicologically relevant (R)-epimers and the biologically inactive (S)-epimers for seven pairs of EAs were determined by a HPLC method coupled with fluorescence detection. Baking cookies resulted in degradation of EAs (2–30 %) and a shift in the epimeric ratio toward the (S)-epimer for all EAs. The applied digestion model led to a selective toxification of ergotamine and ergosine, two ergotamine-type EAs. The initial percentage of the toxic (R)-epimer in relation to the total toxin content was considerably increased after digestion of cookies. Ergotamine and ergosine increased from 32 to 51 % and 35 to 55 %, respectively. In contrast, EAs of the ergotoxine type (ergocornine, α- and β-ergocryptine, and ergocristine) showed an epimeric shift toward their biologically inactive (S)-epimers. Further experiments indicated that the selective epimerization of ergotamine EAs occurs in the duodenal juice only. These results demonstrate that toxification of EAs in the intestinal tract should be taken into consideration.

Keywords: HPLC-FLD; Claviceps purpurea ; Rye; Ergotoxine; Ergotamine

New quality-control materials for the determination of alkylphenols and alkylphenol ethoxylates in sewage sludge by María Fernández-Sanjuan; Silvia Lacorte; Anna Rigol; Angels Sahuquillo (pp. 2499-2505).

The determination of alkylphenols in sewage sludge is still hindered by the complexity of the matrix and of the analytes, some of which are a mixture of isomers. Most of the methods published in the literature have not been validated, due to the lack of reference materials for the determination of alkylphenols in sludge. Given this situation, the objectives of the present study were to develop a new quality-control material for determining octylphenol, nonylphenol and nonylphenol monoethoxylate in sludge. The material was prepared from an anaerobically digested sewage sludge, which was thermally dried, sieved, homogenized and bottled after checking for the bulk homogeneity of the processed material. Together with the sewage sludge, an extract was also prepared, in order to provide a quality-control material for allowing laboratories to test the measuring step. The homogeneity and 1-year stability of the two materials were evaluated. Statistical analysis proved that the materials were homogeneous and stable for at least 1 year stored at different temperatures. These materials are intended to assist in the quality control of the determination of alkylphenols and alkylphenol ethoxylates in sewage sludge. Figure Quality-control sewage sludge material for APs determination

Keywords: Alkylphenols; Alkylphenol ethoxylates; Sewage sludge; Quality control; Stability; Homogeneity

Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip by Ayse Nur Polat; Karsten Kraiczek; Albert J. R. Heck; Reinout Raijmakers; Shabaz Mohammed (pp. 2507-2512).

Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO₂) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible. A method was developed that allows the automated, online, dimethyl labeling and TiO₂ enrichment of phosphopeptides from complex samples on a three-sectioned microfluidic HPLC phosphochip. The method is shown to allow quantification over at least one order of magnitude and provides a robust approach for fully automated online quantification of phosphopeptides.

Keywords: HPLC chip; Microfluidics; Dimethyl labeling; Phosphorylation; Quantification; TiO₂

Determination of nitrite, nitrate, bromide, and iodide in seawater by ion chromatography with UV detection using dilauryldimethylammonium-coated monolithic ODS columns and sodium chloride as an eluent by Kazuaki Ito; Ryosuke Nomura; Takuya Fujii; Masahito Tanaka; Tomoaki Tsumura; Hiroyuki Shibata; Takeshi Hirokawa (pp. 2513-2517).

A method was developed for determination of inorganic anions, including nitrite (NO ₂ ⁻ ), nitrate (NO ₃ ⁻ ), bromide (Br⁻), and iodide (I⁻), in seawater by ion chromatography (IC). The IC system used two dilauryldimethylammonium bromide (DDAB)-coated monolithic ODS columns (50 × 4.6 mm i.d. and 100 × 4.6 mm i.d.) connected in series for separation of the ions. Aqueous NaCl (0.5 mol/L; flow rate, 3 mL/min) containing 5 mmol/L phosphate buffer (pH 5) was used as the eluent, and detection was with a UV detector at 225 nm. The monolithic ODS columns were coated and equilibrated with a 1-mmol/L DDAB solution (in H₂O/methanol, 90:10 v/v). The hydrophilic ions (NO ₂ ⁻ , NO ₃ ⁻ , and Br⁻) were separated within 3 min and the retention time of I⁻ was 16 min. No interferences from matrix ions, such as chloride and sulfate ions, were observed in 35 ‰ artificial seawater. The detection limits were 0.6 μg/L for NO ₂ ⁻ , 1.1 μg/L for NO ₃ ⁻ , 70 μg/L for Br⁻, and 1.6 μg/L for I⁻ with a 200-μL sample injection. The performance of the coated columns was maintained without addition of DDAB in the eluent. The IC system was successfully applied to real seawater samples with recovery rates of 94–108 % for all ions. Figure The hydrophilic ions (NO ₂ ⁻ , NO ₃ ⁻ , and Br⁻) and I⁻ in seawater was determined by a single run using the IC system consisting of two dilauryldimethylammonium bromide (DDAB)-coated monolithic ODS columns (50 × 4.6 mm i.d. and 100 × 4.6 mm i.d.) connected in series, NaCl (0.5 mol/L; flow rate, 3 mL/min) containing 5 mmol/L phosphate buffer (pH 5) as the eluent, and a UV detector (225 nm). No interferences from matrix ions, such as chloride and sulfate ions, were observed in 35 ‰ artificial and real seawaters.

Keywords: Inorganic anions; Seawater; Monolithic ODS column; Dilauryldimethylammonium bromide; NaCl mobile phase; Ultraviolet detection

Erratum to: Trapping cells on a stretchable microwell array for single-cell analysis by Yuli Wang; Pavak Shah; Colleen Phillips; Christopher E. Sims; Nancy L. Allbritton (pp. 2519-2519).

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Analytical and Bioanalytical Chemistry (v.404, #8)