Analytical and Bioanalytical Chemistry (v.406, #5)

is currently an Associate Professor at North Carolina A&T State University (NCAT) in Greensboro. Prior to his current position he was an Assistant Professor at Winston Salem State University (WSSU). He obtained his Ph.D. in analytical chemistry from Baylor University under the supervision of Marianna A. Busch and Kenneth W. Busch. He was a postdoctoral researcher in Isiah Warner’s research group at Louisiana State University. He regularly teaches courses and labs in quantitative and instrumental analysis, general chemistry for health sciences majors, and physical science. He initiated the use of a GILE at WSSU and continues their use at NCAT. His research interests include chiral drug analysis, guest-host inclusion complexation, analytical spectroscopy, environmental remediation, fluorescence detection of molecules of pharmaceutical, clinical, biomedical, forensic, and environmental interest as well as chemometrics and multivariate analysis, and experimental design for instrumental calibration, process optimization, and process control.

Biomedical mass spectrometry by Akira Ishii; Mitsutoshi Setou; Toshimitsu Niwa (1273-1274).
has been a Full Professor of the Department of Legal Medicine and Bioethics, Nagoya University Graduate School of Medicine since 2007. His research interests include the application of mass spectrometric methods to forensic toxicology. He is the author of over 120 peer-reviewed papers, 25 scientific papers, and 15 book chapters. has been full professor of Anatomy and Cell Biology at the Hamamatsu University School of Medicine since 2008. His research interests include development and application of imaging mass spectrometry and systems biology with omics technologies. Dr Setou is the author of over 100 peer-reviewed papers, 150 scientific papers, 10 book chapters, and editor of a book on imaging mass spectrometry. is the President of the Japanese Society for Biomedical Mass Spectrometry.

A critical evaluation of the current state-of-the-art in quantitative imaging mass spectrometry by Shane R. Ellis; Anne L. Bruinen; Ron M. A. Heeren (1275-1289).
Mass spectrometry imaging (MSI) has evolved into a valuable tool across many fields of chemistry, biology, and medicine. However, arguably its greatest disadvantage is the difficulty in acquiring quantitative data regarding the surface concentration of the analyte(s) of interest. These difficulties largely arise from the high dependence of the ion signal on the localized chemical and morphological environment and the difficulties associated with calibrating such signals. The development of quantitative MSI approaches would correspond to a giant leap forward for the field, particularly for the biomedical and pharmaceutical fields, and is thus a highly active area of current research. In this review, we outline the current progress being made in the development and application of quantitative MSI workflows with a focus on biomedical applications. Particular emphasis is placed on the various strategies used for both signal calibration and correcting for various ion suppression effects that are invariably present in any MSI study. In addition, the difficulties in validating quantitative-MSI data on a pixel-by-pixel basis are highlighted. Figure Determining localised surface concentrations with quantitative imaging mass spectrometry
Keywords: Imaging mass spectrometry; Quantification; MALDI; SIMS; LA-ICP-MS; Ambient ionization

Many diseases such as arthritis or atherosclerosis are accompanied by inflammatory processes. Inflammation is characterized by the infiltration of cells such as neutrophilic granulocytes and (a) the release of phospholipases [particularly phospholipase A2 (PLA2)] and (b) the generation of reactive oxygen as well as nitrogen species (ROS and RNS). While PLA2 leads to defined lyso products (lacking one acyl residue), lipid oxidation is characterized by much more complex product patterns, including lipid peroxides, aldehydes (by double bond cleavage), and many others. Nevertheless, oxidation processes are highly important under in vivo conditions because molecules with regulatory functions are generated by the oxidation of lipids and/or free fatty acids. Therefore, lipid oxidation products as well as lysolipids are increasingly assumed to represent important disease (bio)markers. Consequently, there is also increasing interest in methods to characterize these products qualitatively and quantitatively.Mass spectrometry (MS) seems to be the method of choice to study (phospho)lipids changed under inflammatory conditions: nowadays, soft ionization MS methods are regularly used to study oxidative lipid modifications because of their high sensitivities and the tremendous mass resolutions that are achievable by using modern mass spectrometers. However, experimental care is required to be able to detect all relevant products. Although electrospray ionization (ESI) MS is so far most popular, applications of matrix-assisted laser desorption and ionization (MALDI) MS are continuously increasing. This review aims to summarize the so far available data on MS analyses of oxidized lipids as well as lysolipids. In addition to model systems, special attention will be paid to the monitoring of oxidized lipids and lysolipids under in vivo conditions. It is the aim of this review to provide a critical survey of the advantages and drawbacks of the different MS methods, with the focus on MALDI and ESI. Figure Scheme of mass spectrometric analysis to study oxidation and enzyme-modified phospholipids changed under inflammatory conditions
Keywords: Phospholipids; MALDI MS; ESI MS; Reactive oxygen species (inflammation); Oxidation; Phospholipases

Imaging mass spectrometry distinguished the cancer and stromal regions of oral squamous cell carcinoma by visualizing phosphatidylcholine (16:0/16:1) and phosphatidylcholine (18:1/20:4) by Yoshiyuki Uchiyama; Takahiro Hayasaka; Noritaka Masaki; Yoshiko Watanabe; Kazuma Masumoto; Tetsuji Nagata; Fuminori Katou; Mitsutoshi Setou (1307-1316).
Most oral cancers are oral squamous cell carcinoma (OSCC). The anatomical features of OSCC have been histochemically evaluated with hematoxylin and eosin. However, the border between the cancer and stromal regions is unclear and large portions of the cancer and stromal regions are resected in surgery. To reduce the resected area and maintain oral function, a new method of diagnosis is needed. In this study, we tried to clearly distinguish the border on the basis of biomolecule distributions visualized by imaging mass spectrometry (IMS). In the IMS dataset, eleven signals were significantly different in intensity (p < 0.01) between the cancer and stromal regions. Two signals at m/z 770.5 and m/z 846.6 were distributed in each region, and a clear border was revealed. Tandem mass spectrometric (MS/MS) analysis identified these signals as phosphatidylcholine (PC) (16:0/16:1) at m/z 770.5 in the cancer region and PC (18:1/20:4) at m/z 846.6 in the stromal region. Moreover, the distribution of PC species containing arachidonic acid in the stromal region suggests that lymphocytes accumulated in response to the inflammation caused by cancer invasion. In conclusion, the cancer and stromal regions of OSCCs were clearly distinguished by use of these PC species and IMS analysis, and this molecular identification can provide important information to elucidate the mechanism of cancer invasion.
Keywords: Imaging mass spectrometry (IMS); Oral cancer; Phosphatidylcholine; Palmitic acid; Arachidonic acid; Matrix-assisted laser-desorption ionization (MALDI)

The misuse of recombinant human erythropoietin (rhEPO) increases the proliferation/production of erythrocytes, which enhance oxygen transport capacities, and has grave consequences with respect to human health and fairness in sports. For sports drug testing, the current analytical methods for rhEPOs are mainly gel electrophoretic methods, such as isoelectric focusing–polyacrylamide gel electrophoresis. Mass spectrometry is fundamentally necessary for the reliable identification of rhEPOs in doping control. In this study, a high-sensitivity and high-throughput mass spectrometric qualitative detection method for darbepoetin alfa in human urine was established by a bottom-up approach. The novel method involves the immunopurification of human urine (10 mL), protease digestion with endoproteinase Glu-C (V8-protease) in an ammonium bicarbonate buffer (pH 7.8) and ultra-performance liquid chromatography using a charged surface hybrid C18 column coupled with electrospray-ionisation high-sensitivity tandem mass spectrometry for improved selectivity of the target molecules. The specific fragment digested from darbepoetin alfa was 90TLQLHVDKAVSGLRSLTTLLRALGAQKE117 (V11). The lower limit of detection of urinary darbepoetin alfa was 1.2 pg/mL. The limit of detection for the confirmation analysis was estimated to be 5 pg/mL. The developed method allows high-throughput confirmation analysis, namely 6 h for sample preparation and an analytical run time of only 10 min per sample; this high-throughput method dramatically decreases the workload in the laboratory. Darbepoetin alfa could be identified in human urine collected after the intravenous administration of 15 μg darbepoetin alfa (n = 3). This mass spectrometric method is an innovative and powerful tool for detecting darbepoetin alfa in human urine for doping control testing. Figure Product ion mass spectrum of the specific peptide V11 after V8-protease digestion of darbepoetin alfa.
Keywords: Doping; Erythropoietin; Darbepoetin alfa; LC–MS/MS; Mass spectrometry

MALDI-Q-TOF mass spectrometric determination of gold and platinum in tissues using their diethyldithiocarbamate chelate complexes by Kayoko Minakata; Hideki Nozawa; Itaru Yamagishi; Kunio Gonmori; Masako Suzuki; Koutaro Hasegawa; Amin Wurita; Kanako Watanabe; Osamu Suzuki (1331-1338).
A rapid determination method is presented for gold (Au3+) and platinum (Pt4+) in tissues using matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF-MS). Au and Pt ions in wet-ashed tissue solution were reacted with diethyldithiocarbamate (DDC), and the resulting chelate complex ions Au(DDC)2 + and Pt(DDC)3 + were detected by MALDI-Q-TOF-MS using α-cyano-4-hydroxycinnamic acid as a matrix. The limit of detection (LOD) was 0.8 ng/g tissue and the quantification range was 2–400 ng/g for Au, and the LOD was 6 ng/g tissue and the quantification range was 20–4,000 ng/g for Pt. The Pt levels detected by MALDI-Q-TOF-MS in several tissues of a patient overdosed with cisplatin were nearly the same as those detected by flow-injection electrospray ionization mass spectrometry. The LODs of Au and Pt were 0.04 pg per well (sample spot) and 0.3 pg per well, respectively. To our knowledge, this is the first attempt to quantify Au3+ and Pt4+ ions in tissues by MALDI-Q-TOF-MS. A rapid determination method is presented for gold and platinum in tissues using matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry (MALDI-Q-TOF-MS). Au and Pt ions in wet-ashed tissue solution were reacted with diethyldithiocarbamate (DDC), and the resulted chelate complex ions Au(DDC)2 + and Pt(DDC)3 + were detected using α-cyano-4-hydroxycinnamic acid as a matrix.
Keywords: Gold; Platinum; Matrix-assisted laser desorption ionization quadrupole time-of-flight mass spectrometry; α-Cyano-4-hydroxycinnamic acid; Tissue

Metabolic profiling of urine and blood plasma in rat models of drug addiction on the basis of morphine, methamphetamine, and cocaine-induced conditioned place preference by Kei Zaitsu; Izuru Miyawaki; Kiyoko Bando; Hiroshi Horie; Noriaki Shima; Munehiro Katagi; Michiaki Tatsuno; Takeshi Bamba; Takako Sato; Akira Ishii; Hitoshi Tsuchihashi; Koichi Suzuki; Eiichiro Fukusaki (1339-1354).
The metabolic profiles of urine and blood plasma in drug-addicted rat models based on morphine (MOR), methamphetamine (MA), and cocaine (COC)-induced conditioned place preference (CPP) were investigated. Rewarding effects induced by each drug were assessed by use of the CPP model. A mass spectrometry (MS)-based metabolomics approach was applied to urine and plasma of MOR, MA, and COC-addicted rats. In total, 57 metabolites in plasma and 70 metabolites in urine were identified by gas chromatography–MS. The metabolomics approach revealed that amounts of some metabolites, including tricarboxylic acid cycle intermediates, significantly changed in the urine of MOR-addicted rats. This result indicated that disruption of energy metabolism is deeply relevant to MOR addiction. In addition, 3-hydroxybutyric acid, l-tryptophan, cystine, and n-propylamine levels were significantly changed in the plasma of MOR-addicted rats. Lactose, spermidine, and stearic acid levels were significantly changed in the urine of MA-addicted rats. Threonine, cystine, and spermidine levels were significantly increased in the plasma of COC-addicted rats. In conclusion, differences in the metabolic profiles were suggestive of different biological states of MOR, MA, and COC addiction; these may be attributed to the different actions of the drugs on the brain reward circuitry and the resulting adaptation. In addition, the results showed possibility of predict the extent of MOR addiction by metabolic profiling. This is the first study to apply metabolomics to CPP models of drug addiction, and we demonstrated that metabolomics can be a multilateral approach to investigating the mechanism of drug addiction.
Keywords: Metabolomics; Drug addiction; conditioned place preference; Morphine; Methamphetamine; Cocaine

Matrix-assisted laser desorption/ionization imaging mass spectrometry revealed traces of dental problem associated with dental structure by Hirokazu Hirano; Noritaka Masaki; Takahiro Hayasaka; Yoshiko Watanabe; Kazuma Masumoto; Tetsuji Nagata; Fuminori Katou; Mitsutoshi Setou (1355-1363).
Periodontal disease is a serious dental problem because it does not heal naturally and leads to tooth loss. In periodontal disease, inflammation at periodontal tissue is thought as predominant, and its effect against tooth itself remains unclear. In this study, we applied matrix-assisted laser desorption/ionization imaging mass spectrometry (IMS) to teeth for the first time. By comparing anatomical structure of tooth affected with periodontal disease with normal ones, we analyzed traces of the disease on tooth. We found signals characteristic of enamel, dentin, and dental pulp, respectively, in mass spectra obtained from normal teeth. Ion images reconstructed using these signals showed anatomical structures of the tooth clearly. Next, we performed IMS upon teeth of periodontal disease. Overall characteristic of the mass spectrum appeared similar to normal ones. However, ion images reconstructed using signals from the tooth of periodontal disease revealed loss of periodontal ligament visualized together with dental pulp in normal teeth. Moreover, ion image clearly depicted an accumulation of signal at m/z 496.3 at root surface. Such an accumulation that cannot be examined only from mass spectrum was revealed by utilization of IMS. Recent studies about inflammation revealed that the signal at m/z 496.3 reflects lyso-phosphatidylcholine (LPC). Infiltration of the signal is statistically significant, and its intensity profile exhibited the influence has reached deeply into the tooth. This suggests that influence of periodontal disease is not only inflammation of periodontal tissue but also infiltration of LPC to root surface, and therefore, anti-inflammatory treatment is required besides conventional treatments.
Keywords: Imaging mass spectrometry (IMS); Matrix-assisted laser ionization/desorption (MALDI); Dental structure; Periodontal disease; Lyso-phosphatidylcholine (LPC)

Exploration of novel predictive markers in rat plasma of the early stages of chronic renal failure by Toshihiro Kobayashi; Yuriko Matsumura; Toshihiko Ozawa; Hiroyuki Yanai; Atsuo Iwasawa; Toshiaki Kamachi; Kouichi Fujiwara; Noriaki Tanaka; Masahiro Kohno (1365-1376).
To identify blood markers for early stages of chronic kidney disease (CKD), blood samples were collected from rats with adenine-induced CKD over 28 days. Plasma samples were subjected to metabolomic profiling by liquid chromatography-mass spectrometry, followed by multivariate analyses. In addition to already-identified uremic toxins, we found that plasma concentrations of N6-succinyl adenosine, lysophosphatidylethanolamine 20:4, and glycocholic acid were altered, and that these changes during early CKD were more sensitive markers than creatinine concentration, a universal indicator of renal dysfunction. Moreover, the increase in plasma indoxyl sulfate concentration occurred earlier than increases in phenyl sulfate and p-cresol sulfate. These novel metabolites may serve as biomarkers in identifying early stage CKD.
Keywords: Chronic renal failure; Metabolomics; LC/MS; Animal model

Imaging of lipids in rat heart by MALDI-MS with silver nanoparticles by Shelley N. Jackson; Kathrine Baldwin; Ludovic Muller; Virginia M. Womack; J. Albert Schultz; Carey Balaban; Amina S. Woods (1377-1386).
Lipids are a major component of heart tissue and perform several important functions such as energy storage, signaling, and as building blocks of biological membranes. The heart lipidome is quite diverse consisting of glycerophospholipids such as phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), phosphatidylglycerols (PGs), cardiolipins (CLs), and glycerolipids, mainly triacylglycerols (TAGs). In this study, mass spectrometry imaging (MSI) enabled by matrix implantation of ionized silver nanoparticles (AgNP) was used to map several classes of lipids in heart tissue. The use of AgNP matrix implantation was motivated by our previous work showing that implantation doses of only 1014/cm2 of 2 nm gold nanoparticulates into the first 10 nm of the near surface of the tissue enabled detection of most brain lipids (including neutral lipid species such as cerebrosides) more efficiently than traditional organic MALDI matrices. Herein, a similar implantation of 500 eV AgNP across the entire heart tissue section results in a quick, reproducible, solvent-free, uniform matrix concentration of 6 nm AgNP residing near the tissue surface. MALDI-MSI analysis of either positive or negative ions produce high-quality images of several heart lipid species. In negative ion mode, 24 lipid species [16 PEs, 4 PIs, 1 PG, 1 CL, 2 sphingomyelins (SMs)] were imaged. Positive ion images were also obtained from 29 lipid species (10 PCs, 5 PEs, 5 SMs, 9 TAGs) with the TAG species being heavily concentrated in vascular regions of the heart.
Keywords: MALDI; Mass spectrometry imaging; Lipids; Silver nanoparticles

Identification of nitrated tyrosine residues of protein kinase G-Iα by mass spectrometry by Jingshan Lu; Ikuko Yao; Masahito Shimojo; Tayo Katano; Hitoshi Uchida; Mitsutoshi Setou; Seiji Ito (1387-1396).
The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization–time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 μM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.
Keywords: MALDI-TOF MS; NanoLC–MS/MS; 3-Nitrotyrosine; Peroxynitrite; Protein kinase G-Iα

A high-throughput method for GMO multi-detection using a microfluidic dynamic array by Fábio Cristiano Angonesi Brod; Jeroen P. van Dijk; Marleen M. Voorhuijzen; Andréia Zilio Dinon; Luis Henrique S. Guimarães; Ingrid M. J. Scholtens; Ana Carolina Maisonnave Arisi; Esther J. Kok (1397-1410).
The ever-increasing production of genetically modified crops generates a demand for high-throughput DNA-based methods for the enforcement of genetically modified organisms (GMO) labelling requirements. The application of standard real-time PCR will become increasingly costly with the growth of the number of GMOs that is potentially present in an individual sample. The present work presents the results of an innovative approach in genetically modified crops analysis by DNA based methods, which is the use of a microfluidic dynamic array as a high throughput multi-detection system. In order to evaluate the system, six test samples with an increasing degree of complexity were prepared, preamplified and subsequently analysed in the Fluidigm system. Twenty-eight assays targeting different DNA elements, GM events and species-specific reference genes were used in the experiment. The large majority of the assays tested presented expected results. The power of low level detection was assessed and elements present at concentrations as low as 0.06 % were successfully detected. The approach proposed in this work presents the Fluidigm system as a suitable and promising platform for GMO multi-detection.
Keywords: High throughput; GMO analysis; Preamplification; Fluidigm system

Visualization of a protein-protein interaction at a single-molecule level by atomic force microscopy by Klaus Bonazza; Hanspeter Rottensteiner; Birgit K. Seyfried; Gerald Schrenk; Günter Allmaier; Peter L. Turecek; Gernot Friedbacher (1411-1421).
Atomic force microscopy is unmatched in terms of high-resolution imaging under ambient conditions. Over the years, substantial progress has been made using this technique to improve our understanding of biological systems on the nanometer scale, such as visualization of single biomolecules. For monitoring also the interaction between biomolecules, in situ high-speed imaging is making enormous progress. Here, we describe an alternative ex situ imaging method where identical molecules are recorded before and after reaction with a binding partner. Relocation of the identical molecules on the mica surface was thereby achieved by using a nanoscale scratch as marker. The method was successfully applied to study the complex formation between von Willebrand factor (VWF) and factor VIII (FVIII), two essential haemostatic components of human blood. FVIII binding was discernible by an appearance of globular domains appended to the N-terminal large globular domains of VWF. The specificity of the approach could be demonstrated by incubating VWF with FVIII in the presence of a high salt buffer which inhibits the interaction between these two proteins. The results obtained indicate that proteins can maintain their reactivity for subsequent interactions with other molecules when gently immobilized on a solid substrate and subjected to intermittent drying steps. The technique described opens up a new analytical perspective for studying protein-protein interactions as it circumvents some of the obstacles encountered by in situ imaging and other ex situ techniques. Figure Complex formation between VWF and FVIII directly monitored on a mica surface by AFM
Keywords: Atomic force microscopy; Protein morphology; Protein-protein interaction; von Willebrand factor; Factor VIII; Calcium

Proteomic study of a tolerant genotype of durum wheat under salt-stress conditions by Anna Laura Capriotti; Grazia Maria Borrelli; Valentina Colapicchioni; Roberto Papa; Susy Piovesana; Roberto Samperi; Serena Stampachiacchiere; Aldo Laganà (1423-1435).
Salinity is one of the major abiotic stress conditions limiting crop growth and productivity. Duilio is a wheat genotype that shows tolerant behavior in both salt-stress and drought-stress conditions. Toward better understanding of the biochemical response to salinity in this genotype of durum wheat, a comparative label-free shotgun proteomic analysis based on normalized spectral abundance factors was conducted on wheat leaf samples subjected to increasing salt-stress levels (100 and 200 mmol L-1 NaCl) with respect to untreated samples. We found significant changes in 71 proteins for the first stress level, in 83 proteins at the higher salinity level, and in 88 proteins when comparing salt-stress levels with each other. The major changes concerned the proteins involved in primary metabolism and production of energy, followed by those involved in protein metabolism and cellular defense mechanisms. Some indications of different specific physiological and defense mechanisms implicated in increasing tolerance were obtained. The enhanced salinity tolerance in Duilio appeared to be governed by a higher capacity for osmotic homeostasis, a more efficient defense, and an improvement of protection from mechanical stress by increased cell wall lignifications, allowing a better potential for growth recovery.
Keywords: Durum wheat; Shotgun proteomics; Label-free; Salt stress

Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids by Jessica Z. Bereszczak; Marlene Havlik; Victor U. Weiss; Martina Marchetti-Deschmann; Esther van Duijn; Norman R. Watts; Paul T. Wingfield; Guenter Allmaier; Alasdair C. Steven; Albert J. R. Heck (1437-1446).
The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions (‘spikes’) on their surface and are unique in having either T = 3 or T = 4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either near the spike apices (historically the ‘α-determinant’) or in the ‘floor’ regions between them (the ‘β-determinant’). Native mass spectrometry (MS) and gas-phase electrophoretic mobility molecular analysis (GEMMA) were used to monitor the titration of HBV capsids with the antigen-binding domain (Fab) of mAb 3120, which has long defined the β-determinant. Both methods readily distinguished Fab binding to the two capsid morphologies and could provide accurate masses and dimensions for these large immune complexes, which range up to ~8 MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryo-electron microscopy analysis for preliminary characterisation of virus-antibody complexes. Figure Monitoring the binding of the antigen-binding domain (Fab) of mAb 3120 to hepatitis B capsids by native MS and GEMMA
Keywords: Native MS; GEMMA; Hepatitis B; Virus-antibody complexes; Quasi-equivalence; Immune complex

This study demonstrates an untested link between model phenolic compounds and the formation/electrophoretic separation of stable urinary metabolites. Sterically encumbered carbonyl groups were examined, and mass determination was used to confirm the presence and stability of two oxidative metabolites of pentachlorophenol: tetrachloro-1,2-benzoquinone and tetrachloro-1,4-dihydroquinone. Subsequently, baseline resolved separation of pentachlorophenol and the two oxidative metabolites was demonstrated under the following conditions: 75 mM sodium tetraborate buffer (pH = 8.5) with 5 % methanol and 50 mM SDS, +10.0 kV running voltage, injection time = 5.0 s, effective capillary length = 55 cm, and run temperature = 20 °C. Results not only provide key metabolic inferences for pentachlorophenol, they also exhibit improvements in the ability to separate and detect changes in urinary metabolites in response to phenolic-related exposure. Figure Metabolic pathway elucidation towards time- and dose-dependent electrophoretic screening of stable oxidative phenolic compounds. Establishing direct pathways and developing sensitive/selective analytical tools to measure and characterize xenobiotics provides a defined link between potential hazards and suspected health effects.
Keywords: Electrophoretic separation; Urinary metabolites; Phenolic compounds

Metabolomic study in plasma, liver and kidney of mice exposed to inorganic arsenic based on mass spectrometry by M. A. García-Sevillano; M. Contreras-Acuña; T. García-Barrera; F. Navarro; J. L. Gómez-Ariza (1455-1469).
The mechanism of arsenic toxicity still remains unclear, although enzymatic inhibition, impaired antioxidants metabolism and oxidative stress may play a role. The toxicological effects of trivalent inorganic arsenic on laboratory mouse Mus musculus after oral administration (3 mg/kg body weight/day) were investigated along 12 days, using a metabolomic approach based on direct infusion mass spectrometry to polar and lipophilic extracts from different organs and fluids (liver, kidney, and plasma). Positive and negative acquisition modes (ESI+/ESI) were used throughout the experiments. The most significant endogenous metabolites affected by exposure were traced by partial least square-discriminant analysis and confirmed by tandem mass spectrometry (MS/MS) and gas chromatography coupled to MS. In this work, the toxic effect of arsenic has been related with important metabolic pathways, such as energy metabolism (e.g., glycolysis, Krebs cycle), amino acids metabolism, choline metabolism, methionine cycle, and degradation of membrane phospholipids (cell apoptosis). In addition, this work illustrates the high reliability of mass spectrometry based on a metabolomic approach to study the biochemical effects induced by metal exposure. Figure Metabolomic study in plasma, liver and kidney of mice exposed to inorganic arsenic based on mass spectrometry
Keywords: Arsenic; ICP-MS; Metabolomics; Metals; Mus musculus ; Mass spectrometry

A new method for measuring perfluoroalkyl contaminants (PFCs) in biological matrices has been developed. An ultra-high pressure liquid chromatograph equipped with a quadrupole time-of-flight mass spectrometer (UPLC-QToF) was optimized using a continuous precursor/product ion monitoring mode. Unlike traditional targeted studies that isolate precursor/product ion pairs, the current method alternates between two ionization energy channels to continuously capture standard electrospray ionization (low energy) and collision induced dissociation (high energy) spectra. The result is the indiscriminant acquisition of paired low and high energy spectra for all constituents eluting from the chromatographic system. This technique was evaluated for the routine analysis of perfluoroalkyl species. Using this technique, linear perfluoroalkyl carboxylic acids (C4 to C14) and perfluoroalkyl sulfonates (C4, C6, C8 and C10) exhibited a linear range spanning over three orders of magnitude and were detectable at levels less than 1 pg on column with a root mean squared signal to noise ratio of 5 to 20. Lake trout (Salvelinus namaycush) and National Institutes of Standards and Technology Standard Reference Material 1946 were used to evaluate matrix effects and the accuracy of this method when applied to a whole fish extract. The current method was also evaluated as a diagnostic tool to identify unknown PFCs using experimental fragmentation patterns, mass defect filtering and Kendrick plots. Figure The future of toxics analysis in biological media: cataloging spectral fingerprints at targeted analysis sensitivity.
Keywords: PFOS; QToF; MSe ; Lake trout; Perfluorinated sulfonates; Perfluorinated acids

Study of the ESI and APCI interfaces for the UPLC–MS/MS analysis of pesticides in traditional Chinese herbal medicine by Lina Chen; Fengrui Song; Zhiqiang Liu; Zhong Zheng; Junpeng Xing; Shuying Liu (1481-1491).
In this work, 53 selected pesticides of different chemical groups were extracted from Chinese herbal medicines and determined by ultra-high-performance liquid chromatography (UHPLC)–tandem mass spectrometry (MS/MS) using both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). Extracts were obtained using the acetonitrile-based quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation technique. Cleanup was performed by dispersive solid-phase extraction using primary secondary amine, graphitized carbon black, and octadecylsilane. Two atmospheric-pressure interfaces, ESI and APCI, were checked and compared. The validation study, including detection limits, linearity, and matrix effects, was conducted on fritillaria, radix ginseng, folium isatidis, semen persicae, and flos lonicerae in multiple reaction monitoring mode. These matrices represent a variety of plants used in traditional Chinese medicine. Fritillaria and radix ginseng were chosen as representatives for roots, folium isatidis was chosen as a representative for leaves, semen persicae was chosen as a representative for seeds, and flos lonicerae was chosen as a representative for flowers. The limits of detection for pesticides were lower in the UHPLC–ESI-MS/MS method than in the UHPLC–APCI-MS/MS method. Matrix effects on the two ionizations were evaluated for the five matrices. Soft signal enhancement in UHPLC–APCI-MS/MS and signal suppression in UHPLC–ESI-MS/MS were observed. Figure Overview of UPLC–MS/MS assay for comparing the APCI and ESI interfaces
Keywords: Pesticide residues; Liquid chromatography–tandem mass spectrometry; Atmospheric pressure chemical ionization; Electrospray ionization; Chinese herbal medicine

The use of polymer materials in industry for product packaging is increasing. The presence of additives in the polymer matrix enables the modification or improvement of the properties and performance of the polymer, but these industries are concerned regarding the extractability of these additives. The quantification of these additives is particularly challenging because of the presence of these substances as contaminants in all the analytical equipment and the diversity of their physicochemical properties. In this context, a multi-residue analytical method was developed for the trace analysis of the twenty main additives (and their degradation products) authorized in plastic products such as pharmaceutical packaging (e.g., antioxidants, release agents, and light absorbers). This analytical method consisted of a solid phase extraction (SPE) followed by an analysis using ultra-high performance liquid chromatography coupled to a tandem mass spectrometer (UHPLC-MS/MS). A comparison of two ionization interfaces and the optimization of the extraction procedure were discussed. The influence of the quality of the solvent type (distilled versus not distilled) and the nature of the SPE cartridges (Polypropylene versus Teflon®) were demonstrated. The optimized method exhibited a quantification limit lower than 20 ng mL−1 and recoveries between 70 % and 120 % for all compounds. Finally, the method was validated according to the ICH directive and was subsequently applied to the extraction of polymers under different pH conditions and storage temperatures. To the best of our knowledge, this study presents the first methodology allowing the simultaneous quantification of 24 additives at low ng mL−1.
Keywords: UHPLC-MS/MS, ultra-high performance liquid chromatography–tandem mass spectrometry; Migration; Polymer; Plastic; Packaging; Additives

ToF-SIMS analysis of poly(l-lysine)-graft-poly(2-methyl-2-oxazoline) ultrathin adlayers by Bidhari Pidhatika; Yin Chen; Geraldine Coullerez; Sameer Al-Bataineh; Marcus Textor (1509-1517).
Understanding of the interfacial chemistry of ultrathin polymeric adlayers is fundamentally important in the context of establishing quantitative design rules for the fabrication of nonfouling surfaces in various applications such as biomaterials and medical devices. In this study, seven poly(l-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL–PMOXA) copolymers with grafting density (number of PMOXA chains per lysine residue) 0.09, 0.14, 0.19, 0.33, 0.43, 0.56, and 0.77, respectively, were synthesized and characterized by means of nuclear magnetic resonance spectroscopy (NMR). The copolymers were then adsorbed on Nb2O5 surfaces. Optical waveguide lightmode spectroscopy method was used to monitor the surface adsorption in situ of these copolymers and provide information on adlayer masses that were then converted into PLL and PMOXA surface densities. To investigate the relationship between copolymer bulk architecture (as shown by NMR data) and surface coverage as well as surface architecture, time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis was performed. Furthermore, ToF-SIMS method combined with principal component analysis (PCA) was used to verify the protein resistant properties of PLL–PMOXA adlayers, by thorough characterization before and after adlayer exposure to human serum. ToF-SIMS analysis revealed that the chemical composition as well as the architecture of the different PLL–PMOXA adlayers indeed reflects the copolymer bulk composition. ToF-SIMS results also indicated a heterogeneous surface coverage of PLL–PMOXA adlayers with high grafting densities higher than 0.33. In the case of protein resistant surface, PCA results showed clear differences between protein resistant and nonprotein-resistant surfaces. Therefore, ToF-SIMS results combined with PCA confirmed that the PLL–PMOXA adlayer with brush architecture resists protein adsorption. However, low increases of some amino acid signals in ToF-SIMS spectra were detected after the adlayer has been exposed to human serum. Figure ᅟ
Keywords: PLL–PMOXA; Graft copolymer; ToF-SIMS; PCA; Adlayer; Protein resistant

Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models by Emelie Westberg; Ulla Hedebrant; Johanna Haglund; Tomas Alsberg; Johan Eriksson; Albrecht Seidel; Margareta Törnqvist (1519-1530).
Stable adducts to serum albumin (SA) from electrophilic and genotoxic compounds/metabolites can be used as biomarkers for quantification of the corresponding in vivo dose. In the present study, conditions for specific analysis of stable adducts to SA formed from carcinogenic polycyclic aromatic hydrocarbons (PAH) were evaluated in order to achieve a sensitive and reproducible quantitative method. Bulky adducts from diolepoxides (DE) of PAH, primarily DE of benzo[a]pyrene (BPDE) and also DE of dibenzo[a,l]pyrene (DBPDE) and dibenzo[a,h]anthracene (DBADE), were used as model compounds. The alkylated peptides obtained after enzymatic hydrolysis of human SA modified with the different PAHDE were principally PAHDE-His-Pro, PAHDE-His-Pro-Tyr and PAHDE-Lys. Alkaline hydrolysis under optimised conditions gave the BPDE-His as the single analyte of alkylated His, but also indicated degradation of this adduct. It was not possible to obtain the BPDE-His as one analyte from BPDE-alkylated SA through modifications of the enzymatic hydrolysis. The BPDE-His adduct was shown to be stable during the weak acidic conditions used in the isolation of SA. Enrichment by HPLC or SPE, but not butanol extraction, gave good recovery, using Protein LoBind tubes. A simple internal standard (IS) approach using SA modified with other PAHDE as IS was shown to be applicable. A robust analytical procedure based on digestion with pronase, enrichment by HPLC or SPE, and analysis with HPLC/MS-MS electrospray ionisation was achieved. A good reproducibility (coefficient of variation (CV) 11 %) was obtained, and the achieved limit of detection for the studied PAHDE, using standard instrumentation, was approximately 1 fmol adduct/mg SA analysing extract from 5 mg SA. Figure An outline of the method for analysis of bulky SA-adducts. All steps/conditions were evaluated.
Keywords: Bulky serum albumin adducts; Polycyclic aromatic hydrocarbons; Extraction (SPE | HPLC | butanol); Diol epoxides; Mass Spectrometry; Hydrolysis (pronase enzymatic | alkaline)

A solid-phase fluorescent biosensor for the determination of phenolic compounds (simple substituted phenols and catecholamines) and peroxides has been developed. The biosensor has a simple construction and the analytical signal is measured directly in a biosensitive layer {peroxidase-chitosan} on the sensor surface. This approach allowed analyzing samples with complex matrices (including water-insoluble samples and nontransparent solutions) without their preliminary pretreatment. Two novel fluorescent indicator reactions for the determination of the above-mentioned analytes in wide concentration ranges (from nmol l−1 to mm l−1) which provided an analytical signal registration on a solid phase were proposed. The developed sensor was applied successfully for the analysis of urine, cosmetics, pharmaceuticals preparations, etc.
Keywords: Solid-phase fluorescence biosensor; Phenols; Catecholamines; Peroxides

The determination of 256 multiclass pesticides in lavandin essential oil has been performed by liquid chromatography–electrospray ionization tandem mass spectrometry using the scheduled selected reaction monitoring mode available on a quadrupole-linear ion trap mass spectrometer. With the aim of improving the limits of quantification (LOQs) of the target molecules, a sampling step based on evaporation of the essential oil under a nitrogen flow assisted by controlled heating was tested. The LOQs determined in this case were compared with the values obtained with the classic dilution preparation method. With sampling by dilution, 247 pesticides were detected and quantified at low concentration, with 74 % of the pesticides having LOQs of 10 μg L-1 or less. With the evaporation method, a global improvement of the LOQs was observed, with lower LOQs for 92 active substances and LOQs of 10 μg L-1 or less for 82.8 % of the pesticides. Almost twice as many active substances had an LOQ of 1 μg L-1 or less when the evaporation method was used. Some pesticides exhibited poor recovery or high variance caused by volatilization or degradation during the evaporation step. This behavior was evidenced by the case of thiophanate-methyl, which is degraded to carbendazim. Figure Sampling method by dilution or evaporation in the multiresidue determination of pesticides in essential oils by LC/MS
Keywords: Pesticides; Lavandin essential oil; Multiresidue analysis; Liquid chromatography–tandem mass spectrometry; Sample preparation; Scheduled selected reaction monitoring

A method was developed to sensitively determine safranine T in wolfberry by molecularly imprinted solid-phase extraction (MISPE) coupled with high-performance liquid chromatography and laser-induced fluorescence detection (HPLC-LIF). The MISPE capillary monolithic column was prepared by water-bath in situ polymerization, using safranine T, methacrylic acid (MAA), and ethylene dimethacrylate (EDMA) as template, functional monomer, and cross-linker, respectively. The properties of the homemade MISPE capillary monolithic column, including capacity and specificity, were investigated under optimized conditions and the morphologies of inner polymers were characterized by scanning electron microscopy (SEM). The mean recoveries of safranine T in wolfberry ranged from 91.2 % to 92.9 % and the intraday and interday relative standard deviation (RSD) values all ranged from 3.4 % to 4.2 %. Good linearity was obtained over 0.001–1.0 μg mL–1 (r = 0.9999) with a detection limit (S/N = 3) of 0.4 ng g–1. Under the selected conditions, enrichment factors of over 90-fold were obtained and the extraction on the monolithic column effectively cleaned up the wolfberry matrix. The results demonstrated that the proposed MISPE-HPLC-LIF method could be applied to sensitively determine safranine T in wolfberry. Figure SEM images of the monolithic column prepared with different initiation reaction methods: a UV initiation; b water bath; c 5000-folds magnification of b; d 20000-folds magnification of b
Keywords: Safranine T; MIPs; SPE; In situ polymerization; Monolithic capillary column; Wolfberry

Investigation of chondroitin sulfate D and chondroitin sulfate E as novel chiral selectors in capillary electrophoresis by Qi Zhang; Yingxiang Du; Jiaquan Chen; Guangfu Xu; Tao Yu; Xiaoyi Hua; Jinjing Zhang (1557-1566).
Various chiral selectors have been utilized successfully in capillary electrophoresis (CE); however, the number of polysaccharides used as chiral selectors is still small and the mechanism of enantiorecognition has not been fully elucidated. Chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE), belonging to the group of glycosaminoglycans, are linear, sulfated polysaccharides with large mass. In this paper, they were investigated for the first time for their potential as chiral selectors by CE. The effect of buffer composition and pH, chiral selector concentration, and applied voltage were systematically examined and optimized. A variety of drug enantiomers were resolved in the buffer pH range of 2.8–3.4 using 20 mM Tris/H3PO4 buffer with 5.0 % CSD or CSE and 20 kV applied voltage. A central composite design was used to validate the optimized separation parameters and satisfactory uniformity was obtained. As observed, CSE allowed satisfactory separation of the enantiomers of amlodipine, laudanosine, nefopam, sulconazole, and tryptophan methyl ester, as well as partial resolution of citalopram, duloxetine, and propranolol under the optimized conditions. CSD allowed partial or nearly baseline separation of amlodipine, laudanosine, nefopam, and sulconazole. The results indicated that CSE has a better enantiorecognition capability than CSD toward the tested drugs. Figure Chiral separation of various drug enantiomers in CE with CSE (A) and CSD (B) as chiral selectors
Keywords: Capillary electrophoresis; Enantiomeric separation; Chondroitin sulfate D; Chondroitin sulfate E; Chiral selector

A multiple hollow fibre liquid-phase microextraction method for the determination of halogenated solvent residues in olive oil by J. Manso; T. García-Barrera; J. L. Gómez-Ariza; A. G. González (1567-1571).
The present paper describes a method based on the extraction of analytes by multiple hollow fibre liquid-phase microextraction and detection by ion-trap mass spectrometry and electron capture detectors after gas chromatographic separation. The limits of detection are in the range of 0.13–0.67 μg kg−1, five orders of magnitude lower than those reached with the European Commission Official method of analysis, with three orders of magnitude of linear range (from the quantification limits to 400 μg kg−1 for all the analytes) and recoveries in fortified olive oils in the range of 78–104 %. The main advantages of the analytical method are the absence of sample carryover (due to the disposable nature of the membranes), high enrichment factors in the range of 79–488, high throughput and low cost. The repeatability of the analytical method ranged from 8 to 15 % for all the analytes, showing a good performance.
Keywords: Membrane extraction; Hollow fibre; Multiple extraction; Gas chromatography; Electron capture detection; Mass spectrometry; Olive oil

Erratum to: Matrix-assisted laser desorption/ionization imaging mass spectrometry revealed traces of dental problem associated with dental structure by Hirokazu Hirano; Noritaka Masaki; Takahiro Hayasaka; Yoshiko Watanabe; Kazuma Masumoto; Tetsuji Nagata; Fuminori Katou; Mitsutoshi Setou (1573-1573).