Analytical and Bioanalytical Chemistry (v.390, #7)

Successful collaboration and mentoring by Emil Chuck (1677-1679).
is Health Professions Advisor and Term Assistant Professor of Biology at George Mason University, Fairfax, Virginia, USA. In 2007–2008, he also served as interim coordinator for the Undergraduate-Faculty Apprenticeship Program which supports undergraduate creative and scholarly activity. In addition to being an advisor for the AAAS Science Careers online forum, he is the co-chair of the Diversity Committee of the National Postdoctoral Association. He also serves on the Education and Workforce Subcommittee and the Health Information Technology Subcommittee of the non-profit Northern Virginia Technology Council business alliance. He has earned degrees in biomedical engineering (Duke University) and cell biology (Case Western Reserve University).

, Professor, holds the Chair in Environmental Analytical Chemistry at the University of Aberdeen, Scotland, UK. He has published more than 100 papers and has given more than 50 invited and plenary lectures. His scientific interest lies mainly in the interaction of elements with organisms, the identification of their molecular form and their biotransformation in the environment, which leads to the development of new technologies such as element mapping in biological tissues or the determination of unstable metal–biomolecules with mass spectrometry. He has been on numerous Editorial Boards and has served on the International Advisory Board of Analytical and Bioanalytical Chemistry since 2005.

The aim of this paper is to give an overview of analytical data on the identification of selenium compounds in biological samples with relevance for selenium metabolism. Only studies applying the combination of element-specific inductively coupled plasma mass spectrometry as well as molecular electrospray mass spectrometry detection have been included. Hence, selenium compounds are only considered identified if molecular mass spectra obtained by analysis of the authentic biological sample have been provided. Selenium compounds identified in selenium-accumulating plants and yeast are included, as extracts from such plants and yeast have been widely used for examination of the cancer-preventive effect of selenium in cell lines, animal models and human intervention trials. Hence, these selenium compounds are available for absorption and further metabolism. Identification of selenium metabolites in simulated gastric and intestinal juice, intestinal epithelial tissue, liver and urine is described. Hence, selenium metabolites identified in relation to absorption, metabolism and excretion are included.
Keywords: Bioanalytical methods; Biological samples; High-performance liquid chromatography; Mass spectrometry/inductively coupled mass spectrometry; Speciation

Combined detection by inductively coupled mass spectrometry (ICP-MS) for elemental information (quantification) and electrospray ionization mass spectrometry (ESI-MS) for molecular information (identification) by means of splitting of the eluent after chromatographic separation is a suitable means of analysis for unknown and not commercially available arsenic species. Simultaneous parallel ESI-MS and ICP-MS detection was applied to identify possible metabolites during the interaction of arsenobetaine (AsB) with natural zeolites. AsB, mainly produced by freshwater and marine organisms, is known to be a candidate of low toxicity. To estimate the possible toxicological risk originating from AsB in contact with natural and synthetic zeolites, small particles of a naturally occurring zeolite were mixed with an AsB solution. After a contact time of 56 days the degradation of AsB proceeded with different yields in the case of the natural Mexican zeolites. In contrast, no additional components were detected in the control samples. It was possible to clearly identify the degradation products dimethylarsinate (m/z 139) and dimethylarsinoylacetate (m/z 181) by comparison of the peaks monitored by ESI-MS and ICP-MS. In some other cases the unknown arsenic species could not be identified so clearly from their molecular masses.
Keywords: Inductively coupled mass spectrometry; Electrospray ionization mass spectrometry; Arsenobetaine; Degradation; Zeolite

Use of the bromine isotope ratio in HPLC-ICP-MS and HPLC-ESI-MS analysis of a new drug in development by Filip Cuyckens; Lieve I. L. Balcaen; Kenny De Wolf; Bjorn De Samber; Cis Van Looveren; Rob Hurkmans; Frank Vanhaecke (1717-1729).
A combination of inductively coupled plasma mass spectrometry (ICP-MS) and electrospray ionization mass spectrometry (ESI-MS) was deployed for the metabolite profiling and metabolite identification of a new antituberculosis compound (R207910, also known as TMC207) that is currently in drug development. R207910 contains one bromine atom, allowing the detection by ICP-MS. Fluctuations in the Br sensitivity caused by the HPLC gradient were counteracted by the use of species-unspecific isotope dilution. In order to evaluate the method developed, the results obtained were compared with those acquired via radioactivity detection. HPLC-ESI-MS was used for the structural identification of R207910 and its metabolites. The 79Br/81Br isotope ratio is also valuable in the search for metabolites in the complex background of endogenous compounds obtained using HPLC-ESI-MS analyses. Data-dependent scanning using isotope recognition with an ion trap mass spectrometer or processing of Q-Tof data provides HPLC-ICP-MS-like “bromatograms”. The combination of accurate mass measurements and the fragmentation behavior in the MS2 spectra obtained using the Q-Tof Ultima mass spectrometer or MSn spectra acquired using the LTQ-Orbitrap allowed structural characterization of the main metabolites of R207910 in methanolic dog and rat faeces extracts taken 0–24 h post-dose. Figure Analyses of a rat faeces extract taken 0–24 h post-dose: a HPLC-ICP-MS using isotope dilution, b corresponding Br mass flow chromatogram, c radio-HPLC, d Q-Tof ESI-MS TIC, e Q-Tof ESI-MS bromatogram after Br stripping, f LTQ-Orbitrap ESI-MS2 TIC obtained with isotopic-data-dependent scanning
Keywords: HPLC-ICP-MS; HPLC-ESI-MS; Metabolite profiling; Metabolite identification; Bromine; Isotopic-data-dependent scanning

Complementary molecular and elemental detection of speciated thioarsenicals using ESI-MS in combination with a xenon-based collision-cell ICP-MS with application to fortified NIST freeze-dried urine by Jenny L. Ellis; Sean D. Conklin; Christina M. Gallawa; Kevin M. Kubachka; Andrea R. Young; Patricia A. Creed; Joseph A. Caruso; John T. Creed (1731-1737).
The simultaneous detection of arsenic and sulfur in thioarsenicals was achieved using xenon-based collision-cell inductively coupled plasma (ICP) mass spectrometry (MS) in combination with high-performance liquid chromatography. In an attempt to minimize the 16O16O+ interference at m/z 32, both sample introduction and collision-cell experimental parameters were optimized. Low flow rates (0.25 mL/min) and a high methanol concentration (8%) in the mobile phase produced a fourfold decrease in the m/z 32 background. A plasma sampling depth change from 3 to 7 mm produced a twofold decrease in background at m/z 32, with a corresponding fourfold increase in the signal associated with a high ionization surrogate for sulfur. The quadrupole bias and the octopole bias were used as a kinetic energy discriminator between background and analyte ions, but a variety of tuning conditions produced similar (less than twofold change) detection limits for sulfur (32S). A 34-fold improvement in the 32S detection limit was achieved using xenon instead of helium as a collision gas. The optimized xenon-based collision cell ICP mass spectrometer was then used with electrospray ionization MS to provide elemental and molecular-based information for the analysis of a fortified sample of NIST freeze-dried urine. The 3σ detection limits, based on peak height for dimethylthioarsinic acid (DMTA) and trimethylarsine sulfide (TMAS), were 15 and 12 ng/g, respectively. Finally, the peak area reproducibilities (percentage relative standard deviation) of a 5-ppm fortified sample of NIST freeze dried urine for DMTA and TMAS were 7.4 and 5.4%, respectively.
Keywords: Thioarsenicals; Xenon; Collision cell; Inductively coupled plasma mass spectrometry; Electrospray ionization mass spectrometry

The weakest step in the analytical procedure for speciation analysis is extraction from a biological material into an aqueous solution which undergoes HPLC separation and then simultaneous online detection by elemental and molecular mass spectrometry (ICP–MS/ES-MS). This paper describes a study to determine the speciation of arsenic and, in particular, the arsenite phytochelatin complexes in the root from an ornamental garden plant Thunbergia alata exposed to 1 mg As L−1 as arsenate. The approach of formic acid extraction followed by HPLC–ES-MS/ICP–MS identified different AsIII–PC complexes in the extract of this plant and made their quantification via sulfur (m/z 32) and arsenic (m/z 75) possible. Although sulfur sensitivity could be significantly increased when xenon was used as collision gas in ICP–qMS, or when HR-ICP–MS was used in medium resolution, the As:S ratio gave misleading results in the identification of AsIII–PC complexes due to the relatively low resolution of the chromatography system in relation to the variety of As–peptides in plants. Hence only the parallel use of ES-MS/ICP–MS was able to prove the occurrence of such arsenite phytochelatin complexes. Between 55 and 64% of the arsenic was bound to the sulfur of peptides mainly as AsIII(PC2)2, AsIII(PC3) and AsIII(PC4). XANES (X-ray absorption near-edge spectroscopy) measurement, using the freshly exposed plant root directly, confirmed that most of the arsenic is trivalent and binds to S of peptides (53% As–S) while 38% occurred as arsenite and only 9% unchanged as arsenate. EXAFS data confirmed that As–S and As–O bonds occur in the plants. This study confirms, for the first time, that As–peptides can be extracted by formic acid and chromatographically separated on a reversed-phase column without significant decomposition or de-novo synthesis during the extraction step.
Keywords: ICP-MS/ES-MS; HPLC; As-peptides

Mercury in plants or animal tissue is supposed to occur in the form of complexes formed with biologically relevant thiols (biothiols), rather than as free cation. We describe a technique for the separation and molecular identification of mercury and methylmercury complexes derived from their reactions with cysteine (Cys) and glutathione (GS): Hg(Cys)2, Hg(GS)2, MeHgCys, MeHgGS. Complexes were characterised by electrospray mass spectrometry (MS) equipped with an ion trap and the fragmentation pattern of MeHgCys was explained by using MP2 and B3LYP calculations, showing the importance of mercury–amine interactions in the gas phase. Chromatographic baseline separation was performed within 10 min with formic acid as the mobile phase on a reversed-phase column. Detection was done by online simultaneous coupling of ES-MS and inductively coupled plasma MS. When the mercury complexes were spiked in real samples (plant extracts), no perturbation of the separation and detection conditions was observed, suggesting that this method is capable of detecting mercury biothiol complexes in plants. Figure Separation and structural identification of Hg and MeHg biothiols
Keywords: Mercury; Methylmercury; Biothiols; High-performance liquid chromatography; Electrospray mass spectrometry; Inductively coupled plasma mass spectrometry; Conformation modelling

Stain efficiency and MALDI-TOF MS compatibility of seven visible staining procedures by Jian-feng Lin; Qing-xi Chen; Hong-yu Tian; Xia Gao; Mei-lan Yu; Gen-jun Xu; Fu-kun Zhao (1765-1773).
Visible stain is still the most popular protein staining method used in proteomic approaches. However, most published data have been derived from comparisons between visible dyes and fluorescent dyes. In this work, we have focused on seven widely used visible staining procedures—Neuhoff CCB, blue silver, and five silver stains (LKB SN, He SN, Yan SN, Vorum SN, and Blum SN)—and studied their stain efficiencies and MALDI-TOF MS compatibilities on 1-D and 2-D PAGE. It was concluded that blue silver is slightly better in terms of stain efficiency than Neuhoff CCB, but it presented worse MS compatibility. Neuhoff CCB presented better MS compatibility and superior linearity but worse sensitivity than silver stains. Among the five silvering procedures, He SN showed the best MS compatibility and a reasonable staining efficiency; Yan SN lowered the chances of obtaining the protein identity by PMF but gave the best stain efficiency; Vorum SN gave a very clear background and a great contrast, while Blum SN was the worst in this respect. The implications of these results for the selection of a convenient stain are discussed according to specific objectives as well as practical aspects.
Keywords: Mass spectrometry; Proteomics; Silver stain; Two-dimensional gel electrophoresis

Three microbial inhibition-based screening methods, the fast antimicrobial screening test (FAST), the Premi® test, and the kidney inhibition swab (KIS™) test, were evaluated using penicillin G, sulfadimethoxine, oxytetracyline, tylosin, danofloxacin, streptomycin, neomycin, and spectinomycin at a range of fortified concentrations in beef kidney juice and beef serum. Each antibiotic was individually tested simultaneously using the different assays in replicate experiments. Detection threshold concentrations for each analyte in each screening assay were determined for the different matrices. Each assay gave a different detectability profile for the different antibiotics, with the largest differences related to neomycin, which was more sensitively detected by the FAST, and penicillin G, which was detected at lower levels by the Premi® and KIS™ tests. In addition to practical considerations, analysts can use the information presented in this study to evaluate each kit for applicability to their monitoring needs.
Keywords: Fast antimicrobial screening test; Premi®; KIS™; Screening antibiotic; Residues; Beef

Spectrochemical analysis of powder using 355 nm Nd-YAG laser-induced low-pressure plasma by Zener S. Lie; M. Pardede; R. Hedwig; M. M. Suliyanti; Koo Hendrik Kurniawan; Munadi; Yong-Inn Lee; Kiichiro Kagawa; Isamu Hattori; May On Tjia (1781-1787).
The applicability of spectrochemical analysis of minute amounts of powder samples was investigated using an ultraviolet Nd-YAG laser (355 nm) and low-pressure ambient air. A large variety of chemical powder samples of different composition were employed in the experiment. These included a mixture of copper(II) sulfate pentahydrate, zinc sulfide, and chromium(III) sulfate n-hydrate powders, baby powder, cosmetic powders, gold films, zinc supplement tablet, and muds and soils from different areas. The powder samples were prepared by pulverizing the original samples to an average size of around 30 μm in order to trap them in the tiny micro holes created on the surface of the quartz subtarget. It was demonstrated that in all cases studied, good quality spectra were obtained with low background, free from undesirable contamination by the subtarget elements and featuring ppm sensitivity. A further measurement revealed a linear calibration curve with zero intercept. These results clearly show the potential application of this technique for practical qualitative and quantitative spectrochemical analysis of powder samples in various fields of study and investigation.
Keywords: Spectrochemical analysis; Powder sample; Direct powder analysis; UV laser; Low-pressure plasma; Shock wave; Laser-induced plasma spectroscopy

Determination of selenocysteine and selenomethionine in edible animal tissues by 2D size-exclusion reversed-phase HPLC-ICP MS following carbamidomethylation and proteolytic extraction by Katarzyna Bierla; Mihaly Dernovics; Véronique Vacchina; Joanna Szpunar; Gérard Bertin; Ryszard Lobinski (1789-1798).
A method was developed for the simultaneous determination of selenomethionine (SeMet) and selenocysteine (SeCys) in meat (chicken and lamb muscles) and different offal tissues (heart, liver, kidney). The analytical procedure was based on the protein extraction with urea under reducing conditions (dithiothreitol), derivatization of SeCys and SeMet by carbamidomethylation with iodoacetamide (IAM) followed by quantitative proteolysis. The mixture of the derivatized Se-amino acids was purified by size-exclusion liquid chromatography (LC) and analysed by ion-paring reversed-phase HPLC–inductively coupled plasma mass spectroscopy (ICP MS). The quantification of SeCys and SeMet was carried out by the method of standard additions. 77SeMet was used to control the SeMet derivatization efficiency and recovery. The method was validated by the determination of the Se mass balance. The Se-amino acids accounted for 91 ± 8% of the total selenium (mean of 95 samples of seven tissues analysed over a period of 18 months). The method was applied to the discrimination of the contribution of selenoproteins (containing SeCys) and other Se-containing proteins (containing SeMet) in tissues of animals during supplementation studies (dose–effect and tolerance).
Keywords: Selenomethionine; Selenocysteine; Animal tissues; Supplementation; HPLC-ICP MS

Narrow-bore HPLC–ICP–MS for speciation of copper in mutant mouse neonates bearing a defect in Cu metabolism by Takamitsu Miyayama; Yasumitsu Ogra; Yousuke Osima; Kazuo T. Suzuki (1799-1803).
Minute amounts of tissue supernatants from mouse neonates bearing a mutation in the copper (Cu)-transporter gene, Atp7a, were injected into narrow-bore HPLC coupled with an inductively coupled plasma–mass spectrometer (ICP–MS) to examine Cu metabolism. In the 14-day-old mutant neonates, Cu accumulated in the intestine in the metallothionein (MT)-bound form, and mRNA expression of the two MT isoforms was increased. Meanwhile, Cu in the MT-bound form (Cu-MT) was depleted in the liver and mRNA expression decreased in comparison with wild-type mice. These results suggest that Cu is not secreted by intestinal microvillus cells into bloodstream due to the defect of Atp7a, and systemic depletion of Cu occurred. On the other hand, in the kidneys of mutant mice, Cu accumulated in the MT-bound form despite the fact that mRNA expression of the two MT isoforms was low. Part of Cu-MT in microvillus cells may be released into bloodstream at turnover and be preferably taken up by the kidneys. Consequently, the mRNA expression of MT isoforms was not always coincident with the amounts of MT proteins binding Cu, and narrow bore HPLC–ICP–MS used for MT protein determination is a complementary technique to real-time RT-PCR used for MT mRNA determination in Cu speciation.
Keywords: Atp7a; Copper; ICP–MS; Metallothionein; Speciation

Evaluation of a combined fractionation and speciation approach for study of size-based distribution of organotin species on environmental colloids by Stéphane Dubascoux; Julien Heroult; Isabelle Le Hécho; Martine Potin-Gautier; Gaëtane Lespes (1805-1813).
Results relating to the first original application of an analytical approach combining asymmetric flow field-flow fractionation (As-Fl-FFF) with multi-detection and chemical speciation for determination of organotins in a landfill leachate sample are presented. The speciation analysis involved off-line head-space solid-phase microextraction (HS-SPME)–gas chromatography with pulsed-flame photometric detection (GC–PFPD) performed after three consecutive collections of five different fractions of interest from the As-Fl-FFF system and cross-flow part (assumed to be representative of the <10 kDa phase). After 0.45 μm filtration and without preconcentration before fractionation and speciation analysis, limits of detection (LOD) were 4–45 ng (Sn) L−1 in the sample, with relative standard deviations (RSD) of 3–23%. The As-Fl-FFF fractionation of this sample enables characterization of two distinct populations—organic-rich and inorganic colloids with gyration radius up to 120 nm. Total Sn and mono and dibutyltins (MBT and DBT) appear to be distributed over the whole colloidal phase. Tributyl, monomethyl, monooctyl, and diphenyltins (TBT, MMT, MOcT, and DPhT) were also detected. Quantitative speciation analysis performed on the two colloidal populations and in the <10 kDa phase revealed concentrations from 130 ± 10 (MMT) to 560 ± 50 ng (Sn) L−1 (DPhT).
Keywords: Organotin compounds; Field-flow fractionation (FFF); Speciation; Landfill leachates; Colloids

GC×GC-ECD: a promising method for the determination of dioxins and dioxin-like PCBs in food and feed by Peter Haglund; Peter Korytár; Conny Danielsson; Jordí Diaz; Karin Wiberg; Pim Leonards; Udo A. T. Brinkman; Jacob de Boer (1815-1827).
There is a need for cost-efficient alternatives to gas chromatography (GC)–high-resolution mass spectrometry (HRMS) for the analysis of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (PCBs) in food and feed. Comprehensive two-dimensional GC–micro electron capture detection (GC×GC-μECD) was tested and all relevant (according to the World Health Organisation, WHO) PCDD/Fs and PCBs could be separated when using a DB-XLB/LC-50 column combination. Validation tests by two laboratories showed that detectability, repeatability, reproducibility and accuracy of GC×GC-μECD are all statistically consistent with GC-HRMS results. A limit of detection of 0.5 pg WHO PCDD/F tetrachlorodibenzo-p-dioxin equivalency concentration per gram of fish oil was established. The reproducibility was less than 10%, which is below the recommended EU value for reference methods (less than 15%). Injections of vegetable oil extracts spiked with PCBs, polychlorinated naphthalenes and diphenyl ethers at concentrations of 200 ng/g showed no significant impact on the dioxin results, confirming in that way the robustness of the method. The use of GC×GC-μECD as a routine method for food and feed analysis is therefore recommended. However, the data evaluation of low dioxin concentrations is still laborious owing to the need for manual integration. This makes the overall analysis costs higher than those of GC-HRMS. Further developments of software are needed (and expected) to reduce the data evaluation time. Combination of the current method with pressurised liquid extraction with in-cell cleanup will result in further reduction of analysis costs.
Keywords: Analysis; Comprehensive two-dimensional gas chromatography; Electron capture detection; Polychlorinated biphenyls; Polychlorinated dibenzo-p-dioxins and dibenzofurans; Food and feed

Rapid and quantitative DNA analysis of genetic mutations for polycystic kidney disease (PKD) using magnetic/luminescent nanoparticles by Ahjeong Son; Amy Dhirapong; Dosi K. Dosev; Ian M. Kennedy; Robert H. Weiss; Krassimira R. Hristova (1829-1835).
Rapid and accurate detection of genetic mutations based on nanotechnology would provide substantial advances in detection of polycystic kidney disease (PKD), a disease whose current methods of detection are cumbersome due to the large size and duplication of the mutated gene. In this study, a nanotechnology-based DNA assay was developed for detection of SNPs (single nucleotide polymorphisms) in a feline autosomal dominant PKD (ADPKD) model which can readily be adapted to diagnosis of human ADPKD type 1. Europium and terbium phosphors were doped into gadolinium crystal hosts with a magnetic core, providing stable luminescence and the possibility of magnetic manipulations in a solution-based assay. A hybridization-in-solution DNA assay was optimized for feline PKD gene SNP detection using genomic DNA extracted from feline kidney tissue and blood. This assay showed a substantial differentiation between PKD and control specimens. The nanotechnology-based DNA assay is attractive from the viewpoint of rapid availability, simple methodology, and cost reduction for clinical use to detect mutations involved in human ADPKD and other genetic diseases. Figure Schematic diagram of PKD (Polycystic Kidney Disease) SNPs detection assay using feline genomic DNA in magnetic/luminescent nanoparticle-based DNA hybridization
Keywords: Polycystic kidney disease (PKD); DNA; Single nucleotide polymorphisms (SNPs); Nanoparticles; Hybridization-in-solution; Lanthanide oxide

Studies are described on the metabolism and the toxicological analysis of the amphetamine-derived designer drug 4-chloro-2,5-dimethoxyamphetamine (DOC) in rat urine using gas chromatographic-mass spectrometric techniques. The metabolites identified indicated that DOC was metabolized by O-demethylation at position 2 or 5 of the phenyl ring partly followed by glucuronidation and/or sulfation. The authors’ systematic toxicological analysis procedure using full-scan gas chromatography-mass spectrometry after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of an intake of a dose of DOC in rat urine that corresponds to a common drug user’s dose. Assuming similar metabolism, the STA procedure described should be suitable as proof of an intake of DOC in human urine.
Keywords: 4-Chloro-2,5-dimethoxyamphetamine; Designer drug; Metabolism; Gas chromatography-mass spectrometry; Urinalysis

Determination of IGF-I in horse plasma by LC electrospray ionisation mass spectrometry by Marie-Agnes Popot; Adrian R. Woolfitt; Patrice Garcia; Jean-Claude Tabet (1843-1852).
The insulin-like-growth factor (IGF-I) peptide is considered to be the main indirect marker for growth hormone administration (GH) in a horse. Further to a previous investigation on measurement of IGF-I in plasma samples by mass spectrometry, this study focuses on quantitative and qualitative analysis of intact IGF-I in horse plasma. First, protein-transposing software has been developed for IGF-I to facilitate its quantification by HPLC–electrospray–ion-trap mass spectrometry. Second, product-ion scan experiments on IGF-I have been conducted on standard samples, non-fortified equine plasma samples, fortified plasma samples, and equine GH post-administration samples. This “top-down” approach method enables characterisation of fragment ions corresponding to the carboxy terminal end, which can be useful for the confirmation of the presence of IGF-I in plasma samples. Figure Structure of IGF-I and amino acid sequences of IGF-I and R3 IGF-I. Deconvolution mass spectra of the IGF-I and R3 IGF-I mixture
Keywords: Bioanalytical methods; Biological samples; Mass spectrometry

Gas chromatography–mass spectrometry investigations have been carried out for the structural analysis of trimethylsilyl methyl derivatives of keto-deoxy sugars and sialic acids studied under electron ionization. Fragmentation patterns were determined. The three derivatives undergo some common fragmentation pathways. Formation of fragment ions possessing cyclic resonance-stabilized structures was demonstrated. As the sialic acid derivative contained a N-acetyl substituent, some additional fragmentations occurred and were also elucidated. Thus, the mechanism of fragmentation was revealed for these derivatives and new findings concerning some series of fragment ions are presented.
Keywords: Gas chromatography–electron impact mass spectrometry; 5-N-Acetylneuraminic acid; 2-Keto-3-deoxy-d-manno-octulosonic acid; 2-Keto-3-deoxy-d-glycero-d-galactononulosonic acid; High-resolution mass spectrometry; Metastable ion kinetic energies; Linked scans

Outer-membrane proteomic maps and surface-exposed proteins of Legionella pneumophila using cellular fractionation and fluorescent labelling by A. Khemiri; A. Galland; D. Vaudry; P. Chan Tchi Song; H. Vaudry; T. Jouenne; P. Cosette (1861-1871).
Bacterial surface-associated proteins play crucial roles in host–pathogen interactions and pathogenesis. The identification of these proteins represents an important goal of bacterial proteomics for vaccine development, but also for environmental concerns such as microbial biosensing. Here, we developed such an approach for Legionella pneumophila, a bacterium that causes severe pneumonia. We propose a complementary strategy consisting of (1) a fluorescent labelling of surface-exposed proteins in parallel with (2) a fractionation of the outer-membrane protein extract. These two distinct protein populations were subsequently separated using two-dimensional gel electrophoresis and characterised by mass spectrometry. Within these populations, we found proteins which were expected for the compartments studied, but also a great number of proteins never experimentally described, and also a non-negligible fraction of proteins never described in these fractions. These data provided new routes of inspection for transport and host recognition for Legionella pneumophila. In addition, these results on the membranome and surfaceome show that Legionella in the stationary phase of growth possesses the major determinants to infect host cells. Figure Electron micrograph of Legionella pneumophila in stationnary phase of growth
Keywords: Proteomics; Fluorescence; Mass spectrometry; Legionella pneumophila

Analysis of ancient Greco–Roman cosmetic materials using laser desorption ionization and electrospray ionization mass spectrometry by Elsa Van Elslande; Vincent Guérineau; Vincent Thirioux; Ghislaine Richard; Pascale Richardin; Olivier Laprévote; Georges Hussler; Philippe Walter (1873-1879).
Microsamples of pink cosmetic powders from the Greco–Roman period were analyzed using two complementary analytical approaches for identification of the colouring agents (lake pigments originally manufactured from madder plants with an inert binder, usually a metallic salt) present in the samples. The first technique was a methanolic acidic extraction of the archaeological samples with an additional ethyl acetate extraction of the anthraquinone-type colouring agents which were identified using high performance liquid chromatography coupled to electrospray ionization with high resolution mass spectrometry (LC–ESI–HRMS), and the second was direct analysis of a microsample by laser desorption ionization–mass spectrometry (LDI–MS). The latter technique is well suited when the quantity of samples is very low. This soft ionization technique enables the detection of very small quantities of compounds using the combination of positive and negative-ion modes. It was also successfully applied for the direct analysis of some laboratory-made reference compounds. However, the presence of lead in one of these ancient samples induced a spectral suppression phenomenon. In this case and conditional on a sufficient quantity of available sample, the former method is better adapted for the characterization of these anthraquinone-type molecules. This study also confirmed that purpurin, munjistin, and pseudopurpurin are the principal colouring agents present in these ancient cosmetic powders constituted from madder plants.
Keywords: Laser desorption ionization (LDI); Mass spectrometry; LDI–MS; Liquid chromatography–electrospray ionization mass spectrometry; LC–ESI–HRMS; Madder plants; Greco–Roman cosmetics; Anthraquinones

Analysis of human histone H4 by capillary electrophoresis in a pullulan-coated capillary, LC-ESI-MS and MALDI-TOF-MS by Stefano Olmo; Roberto Gotti; Marina Naldi; Vincenza Andrisano; Natalia Calonghi; Carola Parolin; Lanfranco Masotti; Vanni Cavrini (1881-1888).
The object of the present study was the analysis of the human histone H4 (a core histone) in order to evaluate the state of its acetylation. Capillary electrophoresis (CE) using a pullulan-coated capillary provides a rapid and efficient approach to the separation of monoacetylated, diacetylated and triacetylated H4 isoforms from human cells. By using a simple running buffer of 100 mM triethanolamine-phosphate solution at pH 2.5 and exploiting the effectiveness of pullulan-based coverage in preventing adsorptive phenomena, the separation of the differently acetylated isoforms was achieved in less than 15 min with high efficiency and reproducibility. The proposed method was for the first time applied in the analysis of histone H4 fractions obtained from cell lines treated with different histone deacetylase (HDAC) inhibitors, used as potential anticancer drugs. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) analysis demonstrated that the acetylation occurred in the histone H4 tail, whereas the CE separation allowed for a fast determination of the percentages of H4 acetylated isoforms in real samples; the results were in agreement with those obtained from liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) analysis. Therefore, the proposed CE method is a useful complementary support to the hyphenated techniques for the rapid monitoring of the activity of HDAC inhibitors.
Keywords: Capillary electrophoresis; Coated capillary; Histones; LC-ESI-MS; MALDI-TOF-MS

Contribution of multi-nuclear solid state NMR to the characterization of the Thalassiosira pseudonana diatom cell wall by Benoit Tesson; Sylvie Masse; Guillaume Laurent; Jocelyne Maquet; Jacques Livage; Véronique Martin-Jézéquel; Thibaud Coradin (1889-1898).
A major issue in the study of biosilicification processes is the harsh chemical conditions required for silica dissolution, which often lead to denaturation of the associated bio-organic matter. In order to demonstrate the potential of solid state NMR for investigating silicified materials of natural origin, this technique was applied to isotopically enriched Thalassiosira pseudonana diatom cells. 29Si, 1H,31P, 13C and 15N solid state NMR studies were performed on whole cells, SDS-extracted and H2O2-cleaned silica shells. Cross-polarization techniques were useful for identifying the presence of mobile and rigid molecules, allowing loosely bound and silica-entrapped species to be discriminated. Successive cleaning procedures efficiently eliminated weakly associated organic matter. The H2O2-cleaned silica shell still contained carbohydrates (mainly chitin) and proteins as well as lipids. This suggests that the role of lipids in diatom shell formation may have been underestimated so far, demonstrating the potential of solid state NMR for studying composite biomaterials.
Keywords: Diatom; Frustule; Nuclear magnetic resonance; Silica; Thalassiosira pseudonana

A method for the determination of 1-hydroxypyrene in urine and hexachlorbenzene in water applying the regression triplet in the calibration procedure of chromatographic data has been applied. The detection limit and quantification limit are currently calculated on the basis of the standard deviation of replicate analyses at a single concentration. However, since the standard deviation depends on concentration, these single-concentration techniques result in limits that are directly dependent on spiking concentration. A more rigorous approach requires first careful attention to the three components of the regression triplet (data, model, method), examining (1) the data quality of the proposed model, (2) the model quality and (3) the least-squares method to be used for fulfilment of all least-squares assumptions. For high-performance liquid chromatography determination of 1-hydroxypyrene in urine and gas chromatography analysis of hexachlorbenzene in water, this paper describes the effects of deviations from five basic assumptions The paper considers the correction of deviations: identifying influential points, namely, outliers, the calibration task depends on the regression model used, and the least-squares method is based on the assumptions of the normality of the errors, homoscedasticity and the independence of errors. Results show that the approach developed provides improved estimates of analytical limits and that the single-concentration approaches currently in wide use are seriously flawed.
Keywords: Calibration precision; Outliers; Influential points; Iterative reweighted least squares; Detection limit; 1-hydroxypyrene; Hexachlorbenzene

Near infrared spectroscopy as a rapid tool to measure volatile aroma compounds in Riesling wine: possibilities and limits by H. E. Smyth; D. Cozzolino; W. U. Cynkar; R. G. Dambergs; M. Sefton; M. Gishen (1911-1916).
Volatile chemical compounds responsible for the aroma of wine are derived from a number of different biochemical and chemical pathways. These chemical compounds are formed during grape berry metabolism, crushing of the berries, fermentation processes (i.e. yeast and malolactic bacteria) and also from the ageing and storage of wine. Not surprisingly, there are a large number of chemical classes of compounds found in wine which are present at varying concentrations (ng L−1 to mg L−1), exhibit differing potencies, and have a broad range of volatilities and boiling points. The aim of this work was to investigate the potential use of near infrared (NIR) spectroscopy combined with chemometrics as a rapid and low-cost technique to measure volatile compounds in Riesling wines. Samples of commercial Riesling wine were analyzed using an NIR instrument and volatile compounds by gas chromatography (GC) coupled with selected ion monitoring mass spectrometry. Correlation between the NIR and GC data were developed using partial least-squares (PLS) regression with full cross validation (leave one out). Coefficients of determination in cross validation (R 2) and the standard error in cross validation (SECV) were 0.74 (SECV: 313.6 μg L−1) for esters, 0.90 (SECV: 20.9 μg L−1) for monoterpenes and 0.80 (SECV: 1658 μg L−1) for short-chain fatty acids. This study has shown that volatile chemical compounds present in wine can be measured by NIR spectroscopy. Further development with larger data sets will be required to test the predictive ability of the NIR calibration models developed.
Keywords: Volatile chemical compounds; Wines; Partial least squares; Near infrared; Riesling

Major flavonoid components of heartsease (Viola tricolor L.) and their antioxidant activities by Viktoria Vukics; Agnes Kery; Guenther K. Bonn; Andras Guttman (1917-1925).
Sephadex LH-20 column chromatography was used to separate flavonoid components in a heartsease methanol extract. One of the main components was identified by NMR as violanthin (6-C-glucosyl-8-C-rhamnosylapigenin). As a first approximation, the other main flavonoid component was considered to be rutin (3-O-rhamnoglucosylquercetin), based on comprehensive comparison of retention times and UV spectra of reference molecules, as well as molecular mass and fragmentation patterns obtained by mass spectrometry. The minor flavonoids were separated by polyamide column and analyzed by LC-MS. The antioxidant capacity of different flavonoid fractions was determined using both Trolox equivalent antioxidant capacity (TEAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) in vitro antioxidant assays. The highest electron-donor capacity was found for the major flavonoid component (rutin), whereas one minor component-rich flavonoid fraction exhibited the highest hydrogen-donor activity.
Keywords: Flavonoid isolation; Column chromatography; NMR; LC-MS; Antioxidants

Synthesis of chitosan resin possessing a phenylarsonic acid moiety for collection/concentration of uranium and its determination by ICP-AES by Koji Oshita; Kenji Seo; Akhmad Sabarudin; Mitsuko Oshima; Toshio Takayanagi; Shoji Motomizu (1927-1932).
A chitosan resin possessing a phenylarsonic acid moiety (phenylarsonic acid type chitosan resin) was developed for the collection and concentration of trace uranium prior to inductively coupled plasma (ICP) atomic emission spectrometry (AES) measurement. The adsorption behavior of 52 elements was systematically examined by packing it in a minicolumn and measuring the elements in the effluent by ICP mass spectrometry. The resin could adsorb several cationic species by a chelating mechanism, and several oxo acids, such as Ti(IV), V(V), Mo(VI), and W(VI), by an anion-exchange mechanism and/or a chelating mechanism. Especially, U(VI) could be adsorbed almost 100% over a wide pH region from pH 4 to 8. Uranium adsorbed was easily eluted with 1 M nitric acid (10 mL), and the 25-fold preconcentration of uranium was achieved by using a proposed column procedure, which could be applied to the determination of trace uranium in seawater by ICP-AES. The limit of detection was 0.1 ng mL−1 for measurement by ICP-AES coupled with 25-fold column preconcentration.
Keywords: Inductively coupled plasma atomic emission spectrometry; Column procedure; Uranium; Phenylarsonic acid; Chitosan

The present work describes a headspace single-drop microextraction (HS-SDME) method in conjunction with gas chromatography electron capture detection (GC–ECD) for the determination of an organophosphate insecticide, chlorpyrifos-ethyl (CPF), in rat liver. Sample preparation included tissue homogenization with methanol in the presence of anhydrous sodium sulfate in order to isolate CPF from the matrix, followed by dilution with 10 mL of 0.1 M H2SO4 and headspace microextraction to a 2-μL drop of 1-octanol. The main factors affecting extraction efficiency were optimized [temperature 90 °C, preheating and extraction times of eight and six minutes, respectively, 2 g of (NH4)2SO4, stirring rate of 1000 rpm, 200 μL of methanolic extract]. The method allows for the separation and quantitation of residue levels of CPF in the livers of rats exposed orally to that insecticide. Using internal standardization (with chlorpyrifos-methyl used as an internal standard), the linearity of the method was demonstrated in the range 10–2500 ng g−1 with a correlation coefficient R > 0.996 and a satisfactory level of precision (RSD 3.85%, n = 6). Moreover, the results obtained with the new method do not differ from those obtained with the conventional residue method used in our laboratory. The feasibility of this HS-SDME approach as an equivalent analytical method for the determination of CPF in rat liver that possessess advantages such as low cost, low solvent consumption and high throughput was confirmed. Figure Headspace single-drop microextraction
Keywords: Chlorpyrifos-ethyl; Single-drop microextraction; GC–ECD; Organophosphate insecticides

An optical chemical sensor for mercury ion based on 2-mercaptopyrimidine in PVC membrane by Bahareh Khezri; Mohammad K. Amini; Ali R. Firooz (1943-1950).
An optical chemical sensor based on 2-mercaptopyrimidine (2-MP) in plasticized poly(vinyl chloride) (PVC) membrane incorporating (N,N-diethyl-5-(octadecanoylimino)-5H benzo[a]phenoxazine-9-amine (ETH 5294) and sodium tetraphenyl borate (NaTPB) for batch and flow-through determination of mercury ion is described. The response of the sensor is based on selective complexation of Hg2+ with 2-MP in the membrane phase, resulting in an ion exchange process between H+ in the membrane and Hg2+ in the sample solution. The influences of several experimental parameters, such as membrane composition, pH, and type and concentration of the regenerating reagent, were investigated. The sensor has a response range of 2.0 × 10−9 to 2.0 × 10−5 mol L−1 Hg2+ with a detection limit of 4.0 × 10−10 mol L−1 and a response time of ≤45 s at optimum pH of 6.5 with high measurement repeatability and sensor-to-sensor reproducibility. It shows high selectivity for Hg2+ over several transition metal ions, including Ag+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Mn2+, Ni2+, and common alkali and alkaline earth ions such as Na+, K+, Mg2+, Ca2+, and Pb2+. The sensor membrane can be easily regenerated with dilute acid solutions. The sensor has been used for the determination of mercury ion concentration in water samples.
Keywords: 2-Mercaptopyrimidine; Optical chemical sensor; Mercury ion; Trace elements; Water analysis

Adsorptive stripping voltammetry is a very sensitive and selective method for determination of drugs and organic substances in biological fluids. We have shown that determination of testosterone by adsorptive stripping voltammetry is possible using a lead film electrode. The lead film plating process and accumulation of testosterone were performed simultaneously from an acetate buffer solution of pH = 5.2 at a potential of −1.1 V. The measurements were carried out in undeaerated solutions. The detection limit was 9 × 10−9 mol L−1 for an accumulation time of 120 s; the relative standard deviation for 1 × 10−7 mol L−1 testosterone was 3.8%. The proposed voltammetric procedure for determination of testosterone could be applied to its determination in a pharmaceutical preparation and human urine samples directly without any separation steps.
Keywords: Lead film electrode; Stripping analysis; Testosterone; Determination; Urine