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Analytical and Bioanalytical Chemistry (v.391, #1)

Isotopic abundance challenge by Juris Meija (pp. 1-2).

Solution to the birthday chromatography challenge by Juris Meija (pp. 3-5).

Liquid chromatography–mass spectrometry: the long road from instrumental developments to applications by Uwe Karst (pp. 7-8).

received his Ph.D. at the University of Münster, Germany, in 1993. Following postdoctoral studies at the University of Colorado in Boulder, USA, and Habilitation in Münster, Germany, in 1998 he was full professor of chemical analysis at the University of Twente, The Netherlands, from 2001–2005. As of 2005 he is a full professor of analytical chemistry at the University of Münster, Germany. His current research interests are hyphenated techniques, speciation analysis, miniaturization, and derivatization.

Health risks of tattoo colors by Rudolf Vasold; Eva Engel; Burkhard König; Michael Landthaler; Wolfgang Bäumler (pp. 9-13).

studied chemistry at the University of Regensburg and was awarded a doctorate for work on trace analysis of explosives. Since 1993 he has worked at the Institute of Organic Chemistry at the University of Regensburg with the emphasis HPLC and GC. studied chemistry and biology at the University of Regensburg. In 2007 she submitted her thesis “Tattoo pigments in skin: determination and quantitative extraction of red tattoo pigments” and received her doctorate from the University of Regensburg under the direction of Professor Burkhard König. received his doctorate in 1991 from the University of Hamburg under the direction of Professor de Meijere. He continued his scientific education as a postdoctoral fellow with Professor M. A. Bennett, Canberra, and Professor B. M. Trost, Stanford. In 1996 he obtained his “Habilitation” at the University of Braunschweig. Since 1999 he has been full professor of organic chemistry at the University of Regensburg. His current research interest is the development of synthetic receptors for peptide and protein recognition. studied medicine from 1967–1973 at the Ludwig-Maximilians-University of Munich and received his MD in 1973. From 1976–1991 he was senior lecturer and Associate Professor at the Ludwig-Maximilians-University of Munich. He received his venia legendi in 1984 and since 1991 has been head of the Department of Dermatology University of Regensburg, Germany. studied physics at the University of Regensburg and received his master’s degree in 1987. He then worked in the field of optics and lasers and received his PhD in physics 1991. Since then he has been working in the medical faculty and is engaged in the field of laser medicine and photobiology. He received his venia legendi in 2002 and is assistant professor at the Medical Faculty of the University of Regensburg.

Quantification with HPLC–MS/MS for environmental issues: quality assurance and quality assessment by Kai Bester (pp. 15-20).

Comprehensive two-dimensional liquid chromatography coupled with mass spectrometry by Jaroslav Pól; Tuulia Hyötyläinen (pp. 21-31).

In this review, instrumental aspects of comprehensive two-dimensional liquid chromatography coupled with mass spectrometry are presented. The milestones of LC×LC are briefly summarized. Instrument configuration, selection of experimental conditions, the different interfaces used in the system and the current applications of LC×LC–MS systems are described.

Keywords: Comprehensive two-dimensional liquid chromatography; Mass spectrometry

Characterisation of historical organic dyestuffs by liquid chromatography–mass spectrometry by Erwin Rosenberg (pp. 33-57).

This review discusses the characterisation of natural organic dyestuffs of historical interest by liquid chromatography–mass spectrometry. The structures of the most important natural organic dyestuffs traditionally used are presented and discussed from the perspective of their analytical chemical determination. The practical aspects of the determination of this inhomogeneous range of compounds with different structures, such as anthraquinones, flavonoids, indigoids or tannins, are discussed with their implications for sample preparation, liquid chromatographic separation and mass spectrometric detection. The particular focus of this review is the discussion of the mass spectral fragmentation patterns of the different classes of natural organic dyestuffs, which in the ideal case allow the identification of the dyestuff actually used, and thereby provide a key to the better characterisation and understanding of historical objects dyed with natural organic dyestuffs. Figure LC-MS allows characterisation of natural dyestuff constituents: the MS spectrum of alizarin is superimposed over a photo of a textile coloured using this red dye

Keywords: Historical dyestuffs; Liquid chromatography–mass spectrometry; Flavonoids; Anthraquinoides; Indigoids; Tannins

High-performance liquid chromatography–tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites by M. Holčapek; L. Kolářová; M. Nobilis (pp. 59-78).

Applications of tandem mass spectrometry (MS/MS) techniques coupled with high-performance liquid chromatography (HPLC) in the identification and determination of phase I and phase II drug metabolites are reviewed with an emphasis on recent papers published predominantly within the last 6 years (2002–2007) reporting the employment of atmospheric pressure ionization techniques as the most promising approach for a sensitive detection, positive identification and quantitation of metabolites in complex biological matrices. This review is devoted to in vitro and in vivo drug biotransformation in humans and animals. The first step preceding an HPLC-MS bioanalysis consists in the choice of suitable sample preparation procedures (biomatrix sampling, homogenization, internal standard addition, deproteination, centrifugation, extraction). The subsequent step is the right optimization of chromatographic conditions providing the required separation selectivity, analysis time and also good compatibility with the MS detection. This is usually not accessible without the employment of the parent drug and synthesized or isolated chemical standards of expected phase I and sometimes also phase II metabolites. The incorporation of additional detectors (photodiode-array UV, fluorescence, polarimetric and others) between the HPLC and MS instruments can result in valuable analytical information supplementing MS results. The relation among the structural changes caused by metabolic reactions and corresponding shifts in the retention behavior in reversed-phase systems is discussed as supporting information for identification of the metabolite. The first and basic step in the interpretation of mass spectra is always the molecular weight (MW) determination based on the presence of protonated molecules [M+H]⁺ and sometimes adducts with ammonium or alkali-metal ions, observed in the positive-ion full-scan mass spectra. The MW determination can be confirmed by the [M-H]^- ion for metabolites providing a signal in negative-ion mass spectra. MS/MS is a worthy tool for further structural characterization because of the occurrence of characteristic fragment ions, either MS n analysis for studying the fragmentation patterns using trap-based analyzers or high mass accuracy measurements for elemental composition determination using time of flight based or Fourier transform mass analyzers. The correlation between typical functional groups found in phase I and phase II drug metabolites and corresponding neutral losses is generalized and illustrated for selected examples. The choice of a suitable ionization technique and polarity mode in relation to the metabolite structure is discussed as well.

Keywords: Metabolite; Drug; Xenobiotic; High-performance liquid chromatography–mass spectrometry; Mass spectrometry; Atmospheric pressure ionization

Biomimetic modeling of oxidative drug metabolism by Wiebke Lohmann; Uwe Karst (pp. 79-96).

The prediction of drug metabolism is an important task in drug development. Besides well-established in vitro and in vivo methods using biological matrices, several biomimetic models have been developed. This review summarizes three different nonenzymatic strategies, including metalloporphyrins as surrogates of the active centre of cytochrome P450, Fenton’s reagent, and the electrochemical oxidation of drug compounds. Although none of the systems can simulate the whole range of cytochrome P450-catalyzed reactions adequately, the biomimetic models show some advantages over standard in vitro methods. For example, metalloporpyhrin catalysts allow the synthesis of certain metabolites in sufficient amounts and with sufficient purities to permit characterization and further pharmacological and toxicological tests. The electrochemical generation of metabolites coupled on-line to liquid chromatography/mass spectrometry is a promising tool for studying reactive metabolites and can be applied in automated high-throughput screening approaches. In this paper, detailed comparisons with cytochrome P450 catalysis are drawn, advantages and disadvantages of the respective methods are revealed, and possible applications are discussed.

Keywords: Drug metabolism; Biomimetic oxidations; Metalloporphyrins; Fenton’s reagent; Electrochemistry/LC/MS

LC-MS-based procedures for monitoring of toxic organophosphorus compounds and verification of pesticide and nerve agent poisoning by Harald John; Franz Worek; Horst Thiermann (pp. 97-116).

Organophosphorus compounds (OPCs) are used worldwide as, e.g., flame retardants, plasticizers, and pesticides and remaining stockpiles of OPC nerve agents are present in military arsenals. These OPCs exhibit acute and potential chronic toxicity to man, the environment, and biota thus emphasizing the need for efficient analytical procedures to monitor potential risk to health. Therefore, this review discusses LC-MS-based procedures for OPC detection, addressing sample preparation, separation, ionization, and detection in comprehensive detail. For sample preparation conventional liquid-liquid extraction (LLE) and diverse solid-phase extraction (SPE) procedures are still used most frequently. Nevertheless, during the last three years a number of sophisticated novel methods have been introduced. Solid-phase microextraction (SPME), stir-bar-sorptive extraction (SBSE), membrane-assisted solvent extraction (MASE), and specifically designed molecularly imprinted polymers (MIP) exhibit high potential for frequent use in the future. Additional emphasis in this review is dedicated to the quite young history and current progress in ionization and MS detection of OPCs. The number of relevant published LC-MS reports has tripled in the last five years. This is especially due to the proliferating use of electrospray ionization (ESI), nowadays an indispensable and reliable tool for LC-MS coupling. LC-MS is becoming an appropriate complementary or replacement method for the more traditional GC-MS methods, and not only for non-volatile, hydrophilic, and ionic OPCs. The last section of this review covers recent approaches for verification of OPC poisoning. LC-MS-MS detection of phosphylated peptides generated from inhibited circulating serum butyrylcholinesterase (BChE) by valuable proteomics techniques enables proof of intoxication on the molecular level. Therefore, this review gives a comprehensive overview on the status quo of LC-MS-based OPC analysis in respect of both technical progress and relevant applications. Figure Monitoring and verification

Keywords: Biomonitoring; Flame retardants; LC-MS; Nerve agents; Organophosphorus compounds; Pesticides; Verification

Determination of marine biotoxins relevant for regulations: from the mouse bioassay to coupled LC-MS methods by Bernd Christian; Bernd Luckas (pp. 117-134).

The frequency of occurrence and intensity of harmful algal blooms (HABs) appear to be increasing on a global scale. Consequently, methods were established for the evaluation of possible hazards caused by the enrichment of algal toxins in the marine food chain. Different clinical types of algae-related poisoning have attracted scientific attention: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). In several countries fish specialties are consumed which may be contaminated with algal toxins typical for the respective region (e.g., ciguatera and tetrodotoxins). Bioassays are common methods for the determination of marine biotoxins. However, biological tests are not completely satisfactory, due to the low sensitivity and the absence of specialized variations. Moreover, there is growing resistance against the use of animal experiments. Therefore, many efforts have been made to determine algal toxins with chemical methods. In this context LC-MS methods replaced HPLC methods with optical detectors, allowing both effective seafood control and monitoring of phytoplankton in terms of the different groups of marine biotoxins.

Keywords: Marine biotoxins; LC-MS/MS; PSP toxins; DSP toxins; Domoic acid; Tetrodotoxins; Ciguatera

On the use of different mass spectrometric techniques for characterization of sequence variability in genomic DNA by Herbert Oberacher (pp. 135-149).

After completion of the human genome sequence the search for differences among individual genomes has become the centre of focus for geneticists. Two different types of polymorphism—single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs)—are major sources of genetic diversity and are of widespread use in genetic analysis. A plethora of genotyping techniques have been developed, and mass spectrometry (MS) is among the most widely used analytical platforms. The most striking advantage of mass spectrometric genotyping assays over others is the use of the measured molecular mass information for allele calling. The molecular mass is less error-prone than other sequence-specific parameters, including migration times, retention times, or hybridization yields, as it represents an intrinsic property of a nucleic acid molecule that is directly related to its nucleotide composition. Mass spectrometric assays can roughly be divided into two major groups—matrix-assisted laser desorption/ionization (MALDI)-based and electrospray ionization (ESI)-based assays. An important subdivision of ESI-based genotyping methods are approaches that originate from the hyphenation of liquid chromatography (LC) to MS. The principles of these three classes of mass spectrometric genotyping techniques are summarized in this review. Possibilities and limitations are critically discussed to assist scientists, especially non-experts in MS, in choosing the appropriate mass spectrometric assay for genotyping a genetic marker of interest. Figure Comparison of the principle workflows applied for the characterization of genetic markers by MALDI–MS, ESI–MS, and LC–MS

Keywords: Review; Mass spectrometry; Liquid chromatography; Electrospray ionization; Matrix-assisted laser desorption/ionization; Genotyping; Nucleic acids; Single nucleotide polymorphism; Short tandem repeat

Hydrophilic interaction liquid chromatography (HILIC) in proteomics by Paul J. Boersema; Shabaz Mohammed; Albert J. R. Heck (pp. 151-159).

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications. Figure Artistic impression of the HILIC separation mechanism

Keywords: HILIC; Proteomics; Post-translational modification; Two-dimensional liquid chromatography

Analysis of nitrophenols in cloud water with a miniaturized light-phase rotary perforator and HPLC-MS by Diana Hofmann; Franziska Hartmann; Hartmut Herrmann (pp. 161-169).

An all-glass miniaturized light-phase rotary perforator for the enrichment of polar compounds has been modified/miniaturized and applied. Its application is demonstrated here for the analysis of nitrophenols and dinitrophenols from low-concentration/low-volume samples. For the method development of high-performance liquid chromatography–mass spectrometry (MS) four eluents were tested: (1) water–methanol, (2) acetic acid–methanol, (3) trifluoroacetic acid–methanol and (4) water–acetonitrile. The last eluent mentioned was used for the subsequent investigation of samples from field experiments. Detection limits varied between 1 ng and 50 pg. The relative standard deviation in repeated measurements was below 15%, corresponding to a good reproducibility. Recoveries ranged between 31 and 100%, showing a significant dependence on the extraction time and the final volume of the sample after evaporation. Quantification was carried out by using deuterated 4-nitrophenol and 2,4-dinitrophenol as standards and applying previously determined response factors. Structure determination of further substances under atmospheric pressure chemical ionization was performed by a first screening with a source collision-induced dissociation, followed by the definite analysis by MS n. The first results are shown for cloud water, fog water and rainwater samples from different locations.

Keywords: High-performance liquid chromatography–mass spectrometry; Liquid–liquid extraction; Nitroaromatics; Phenols; Sample enrichment; Water

Characterization of selected organic compound classes in secondary organic aerosol from biogenic VOCs by HPLC/MS n by Marc-Christopher Reinnig; Lars Müller; Jörg Warnke; Thorsten Hoffmann (pp. 171-182).

The formation of secondary organic aerosols (SOA) has been investigated intensively during the last two decades in numerous field and laboratory studies and a general understanding exists about the major particle-phase products. However, recent studies show that several new product classes, such as esters, peroxides or organosulfates, also have to be considered in order to understand the detailed chemical mechanisms leading to SOA as well as to predict the aerosol mass loadings. For the identification and quantification of these three compound classes as well as for carboxylic SOA compounds, liquid chromatography (LC)/mass spectrometry (MS) is the most appropriate analytical method. In this article we try to summarize briefly the work that has been done for the determination of SOA-related carboxylic acids and we present new LC/tandem MS results on the characterization of esters, peroxides and organosulfates. In contrast to earlier work, the mass-spectrometric characterization of the individual compounds is always based on the comparison with authentic reference compounds.

Keywords: High-performance liquid chromatography; Mass spectrometry; Secondary organic aerosol; Organic acids; Esters; Organic peroxides; Organosulfates

Fast multiresidue screening of 300 pesticides in water for human consumption by LC-MS/MS by Kerstin Greulich; Lutz Alder (pp. 183-197).

The study tested the determination of 300 pesticides in mineral water at levels of 0.1 and 1.0 μg/L. Measurements were conducted by direct sample injection into a liquid chromatograph coupled to a tandem mass spectrometer without any sample enrichment and/or cleanup. Two separate injections enabled the recording of two transitions per analyte (600 selected reaction monitoring transitions in total). For 285 analytes the sensitivity of direct sample injection (100 μL) was sufficient to quantify residues at 0.1 μg/L. All remaining pesticides were detected at 1.0 μg/L. Calibration functions were linear for more than 80% of analytes. Signal suppression or enhancement compared with signals in high-performance liquid chromatography water was equal to or smaller than 20% for 240 analytes. Even the largest matrix-induced suppression did not result in the disappearance of peaks. Combining the results of seven mineral waters, the relative standard deviation of “recovery” was 20% or less for 87% of the substances. A second transition for confirmatory purposes was often available. Consequently, the proposed direct injection of samples without any sample enrichment and/or cleanup is suitable for screening of many pesticides in mineral and drinking water.

Keywords: Liquid chromatography; Tandem mass spectrometry; Electrospray ionization; Drinking water; Pesticide; Residue

Multiresidue analysis of beta-agonists in bovine and porcine urine, feed and hair using liquid chromatography electrospray ionisation tandem mass spectrometry by M. W. F. Nielen; J. J. P. Lasaroms; M. L. Essers; J. E. Oosterink; T. Meijer; M. B. Sanders; T. Zuidema; A. A. M. Stolker (pp. 199-210).

The use of β-agonists as growth promoters in cattle breeding is forbidden in many countries for reasons of fair trade and consumer protection. In recent years the use of liquid chromatography (LC) tandem mass spectrometry (MS/MS) has been shown to be the method of choice for the control of β-agonists. In this study an LC-MS/MS multiresidue analysis method is presented for trace analysis of 22 β-agonists. A truly generic concept has been designed based on mixed-mode solid-phase extraction and positive electrospray ionisation LC-MS/MS operated in the multiple reaction monitoring mode. This method allows application to a wide variety of sample matrices such as urine, feed and hair, following minor modifications to the analysis procedure only. The method features fit-for-purpose sensitivity in urine as shown by CCα and CCβ values of less than 0.2 and less than 0.5 μg/l respectively, for all β-agonists studied (terbutaline and reproterol, less than 0.3 and less than 1.0 respectively). Similar but semiquantitative application to feed and hair showed CCβ values of less than 10.0 and less than 5.0 μg/kg, respectively. A further simplification and improvement is demonstrated using Ultra Performance LC (UPLC™) and fast-switching MS/MS. The successful validation of this method following the latest EU requirements and its application to real samples demonstrate that a new versatile tool has been achieved for veterinary control of β-agonists.

Keywords: Liquid chromatography; Mass spectrometry; Beta-agonists; Feed; Urine; Hair

Folate content in sea buckthorn berries and related products (Hippophaë rhamnoides L. ssp. rhamnoides): LC-MS/MS determination of folate vitamer stability influenced by processing and storage assessed by stable isotope dilution assay by Derek Gutzeit; Sabine Mönch; Gerold Jerz; Peter Winterhalter; Michael Rychlik (pp. 211-219).

A stable isotope dilution assay was adopted for quantitation of folate vitamers in sea buckthorn berries, juice, and concentrate using fourfold labeled folate isotopologues of the folate derivatives as the internal standards and reversed-phase liquid chromatography–tandem mass spectrometry with electrospray ionization (LC-ESI-MS/MS). Processing effects and storage stability were investigated during juice and concentrate production from sea buckthorn berries (Hippophaë rhamnoides). The technological processing of the berries caused a total degradation of tetrahydrofolate and 5-formyltetrahydrofolate in the generated juice. The content of the main folate vitamer 5-methyltetrahydrofolate remained approximately unchanged during the whole processing from the berries to the concentrate. Sea buckthorn juice was stored under two household storage conditions (6 °C, 25 °C), and also under accelerated aging conditions (40 °C) for up to 7 days to determine the effects of storage temperature on the stability of 5-methyltetrahydrofolate. The content of 5-methyltetrahydrofolate was nearly unchanged during the storage at 6 °C after 7 days. The juice showed almost identical degradation of 5-methyltetrahydrofolate of about 17–20% at 25 °C and 40 °C after 7 days of storage. Figure LC-ESI-MS/MS chromatogram in positive ionization mode of a folate extract of seabuckthorn juice. Selected reaction monitoring (SRM) traces of folate vitamers and theirisotopologues: m/z precursor ion/m/z product ion

Keywords: Folate; Stable isotope dilution assay; LC-ESI-MS/MS; Hippophaë rhamnoides ; Sea buckthorn products; Process and storage stability

Profiling of Alk(en)ylresorcinols in cereals by HPLC-DAD-APcI-MS n by Matthias Knödler; Andrea Kaiser; Reinhold Carle; Andreas Schieber (pp. 221-228).

5-Alk(en)ylresorcinols in rye, wheat, spelt, and barley have been characterized by high-performance liquid chromatography coupled to atmospheric pressure chemical ionization multistage mass spectrometry (HPLC-APcI-MS n) for the first time. Among the 29 compounds analysed, several major and minor C₁₅, C₁₇, C₁₉, C₂₁, C₂₃, and C₂₅-substituted resorcinols with saturated, monoenoic, dienoic, and/or oxygenated side-chains were characterized by their specific fragmentation patterns in collision-induced dissociation experiments. Additionally, a C_27:0 homologue, which has probably been overlooked in previous studies based on HPLC alone, was detected in all cereals analysed. Furthermore, we provide tentative evidence for the occurrence of alkylresorcinols with triolefinic side-chains, which have, to our knowledge, so far not been reported in any cereal species.

Keywords: Secale cereale L.; Triticum aestivum L.; Triticum spelta L.; Hordeum vulgare L.; Alk(en)ylresorcinols; HPLC-DAD-APcI-MS n

Liquid chromatography–tandem mass spectrometric method for the determination of salivary 25-hydroxyvitamin D₃: a noninvasive tool for the assessment of vitamin D status by Tatsuya Higashi; Yujin Shibayama; Mihoko Fuji; Kazutake Shimada (pp. 229-238).

A sensitive liquid chromatography–electrospray ionization–tandem mass spectrometric (LC–ESI–MS/MS) method for the determination of 25-hydroxyvitamin D₃ [25(OH)D₃] in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with a Cookson-type reagent, 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), and subjected to LC–MS/MS. The PTAD derivative was much more easily ionized in positive-ESI–MS and efficiently produced a characteristic product ion during MS/MS, compared to the intact 25(OH)D₃. Methylamine was used as the mobile phase additive, and also effectively enhanced the assay sensitivity. Quantification was based on selected reaction monitoring, and 25-hydroxyvitamin D₄ was used as the internal standard. This method allowed the reproducible and accurate quantification of salivary 25(OH)D₃ using a 1.0-ml sample, and the limit of quantitation for 25(OH)D₃ was 2.0 pg/ml. The applicability of the developed method for clinical studies was then examined. There was a positive linear relationship (r ² = 0.830) between the serum 25(OH)D₃ level, which is conventionally used as a means of assessing the vitamin D status, and the salivary 25(OH)D₃ level measured using the proposed method. The method also enabled the detection of the increase in the salivary 25(OH)D₃ level after the supplementation of vitamin D₃.

Keywords: 25-Hydroxyvitamin D₃ ; Saliva; Liquid chromatography–electrospray ionization–tandem mass spectrometry; Derivatization; Mobile phase additive; Cookson-type reagent

Application of LC and GC hyphenated with mass spectrometry as tool for characterization of unknown derivatives of isoflavonoids by Ronald Maul; Nils Helge Schebb; Sabine E. Kulling (pp. 239-250).

Polyphenols belonging to the class of secondary metabolites of plants and microorganisms play an important role as bioactive food constituents as well as contaminants. Structure elucidation of polyphenols in plant extracts or polyphenol metabolites, especially those arising during biotransformation, still represents a challenge for analytical chemistry. Various approaches have been proposed to utilize fragmentation reactions in connection with mass spectrometry (MS) for structural considerations on polyphenolic targets. We compiled and applied specific liquid chromatography (LC)–electrospray ionization in positive mode [ESI(+)]–tandem MS (MS/MS) and gas chromatography (GC)–(electron impact, EI)–MS/MS fragmentation reactions with a special focus on the analysis of isoflavones, whereby this technique was also found to be extendable to determine further polyphenols. For ESI(+)-MS the basic retro-Diels–Alder (rDA) fragmentation offers information about the substitution pattern in the A- and B-rings of flavonoids and the elimination of a protonated 4-methylenecyclohexa-2,5-dienone (m/z = 107) fragment can be used as a diagnostic tool for many isoflavanones. For GC-(EI)-MS/MS analysis after derivatization of the analytes to their trimethylsilyl ethers, the elimination of methyl radicals, tetramethylsilane groups or the combined loss of two methyl groups can be shown to be specific for certain substitution patterns in polyphenols. The applicability of the fragmentation reactions presented is demonstrated exemplarily for three derivatives of the isoflavone irilone. With the help of these fragmentation reactions of the two MS techniques combined, a reliable identification of polyphenols is possible. Especially in such cases where NMR cannot be utilized owing to low analyte amounts being available or prior to purification, valuable information can be obtained.

Keywords: Fragmentation reactions; Gas chromatography–mass spectrometry; Irilone; Isoflavonoides; Liquid chromatography–mass spectrometry; Structure analyses

Determination of benzimidazole- and bicyclic hydantoin-derived selective androgen receptor antagonists and agonists in human urine using LC–MS/MS by Mario Thevis; Maxie Kohler; Andreas Thomas; Joachim Maurer; Nils Schlörer; Matthias Kamber; Wilhelm Schänzer (pp. 251-261).

Selective androgen receptor modulators (SARMs) represent a novel class of drugs with tissue-specific agonistic and antagonistic properties, which are prohibited in sports from January 2008 according to the World Anti-Doping Agency. Preventive approaches to restrict the use of SARMs include early implementation of target analytes into doping control screening assays. Five model SARMs were synthesized, four of which are analogs to prostate-specific androgen receptor antagonists with a 5,6-dichloro-benzimidazole nucleus. The fifth SARM is a muscle-tissue specific agonist with a bicyclic hydantoin structure (BMS-564929). Dissociation pathways after negative electrospray ionization were studied using an LTQ-Orbitrap mass analyzer, and diagnostic product ions and common fragmentation patterns were employed to establish a screening procedure that target the intact SARMs as well as putative metabolic products. Sample preparation based on solid-phase extraction and subsequent LC–MS/MS measurement allowed for detection limits of 1–20 ng/mL, intra- and interday precisions of between 2.4 and 13.2% and between 6.5 and 24.2%, respectively. Recoveries varied from 89 to 106%, and tests for ion suppression or enhancement effects were negative for all analytes. Figure Precursor ion scanning as a tool to screen for the common nucleus of benzimidazole-derived SARMs

Keywords: Doping; Sport; Mass spectrometry; Precursor ion scan; SARMs; Fragmentation; Liquid chromatography

A fast liquid chromatographic tandem mass spectrometric method for the simultaneous determination of total homocysteine and methylmalonic acid by Christel Hempen; Harry Wanschers; Gertjan van der Sluijs Veer (pp. 263-270).

A liquid chromatography(LC)/electrospray ionization tandem mass spectrometry (MS) method for the quantitative determination of total homocysteine and methylmalonic acid and the monitoring of methionine, homocystine and succinic acid in plasma has been developed. The analytes are determined under the presence of the deuterated internal standards methylmalonic acid-d ₃ and homocystine-d ₈. Although methylmalonic acid can be determined directly, a reduction step has to be carried out to ensure the measurement of total homocysteine. Ultrafiltration was applied afterwards to deproteinize the samples prior to LC/MS injection. LC/MS analysis is carried out isocratically using a mobile phase consisting of 5% methanol and 95% of a 0.06 M formic acid solution on a reversed-phase C18 column at a flow rate of 0.5 mL/min. The MS measurement was separated into several periods: homocysteine, homocystine and methionine were determined in the positive-ion mode, whereas the determinations of methylmalonic acid and succinic acid were carried out in the negative-ion mode. The intraday coefficients of variation (CVs) were 2.9% or less and 3.2% or less for homocysteine and methylmalonic acid, respectively. Interday CVs ranged from 3.8 to 5.9% for homocysteine and from 3.5 to 6.3% for methylmalonic acid. Analyte concentrations could reliably be determined, also far below the reference values. Furthermore, the linearity was determined and a correlation study with respect to the existing homocysteine and methylmalonic acid methods at Medisch Spectrum Twente Hospital was carried out.

Keywords: Homocysteine; Methylmalonic acid; Liquid chromatography/mass spectrometry; Tris(2-carboxyethyl)phosphine hydrochloride; Vitamin B₁₂

Liquid chromatography–electrospray tandem mass spectrometry investigations of fragmentation pathways of biliary 4,4′-methylenedianiline conjugates produced in rats by Kan Chen; Tammy R. Dugas; Richard B. Cole (pp. 271-278).

4,4′-methylenedianiline (DAPM) is the main building block for production of 4,4′-methylenediphenyldiisocyanate that has been widely used in the manufacturing of polyurethane materials including medical devices. Although it was revealed that damage to biliary epithelial cells of the liver and common bile duct occurred upon acute exposure to DAPM, the exact mechanism of DAPM toxicity is not fully understood. Both phase I and II biotransformations of DAPM, some of which generate reactive intermediates, are characterized in detail by liquid chromatography electrospray tandem mass spectrometry. The two most prominent metabolites found in rat bile (M2 and M7) implicated glutathione, glucuronic acid, and glycine conjugations (phase II) following hydroxylation, and N-oxidation (phase I). Their decomposition pathways, as evidenced by MS n experiments, have been elucidated in detail. Figure Proposed fragmentation pathways of a DAPM metabolite

Keywords: Methylenedianiline; Biliary metabolites; Glutathione conjugation; Glycine conjugation; Glucuronidation

Detection of low-abundance impurities in synthetic thyroid hormones by stationary phase optimized liquid chromatography–mass spectrometry by Iris Gostomski; Ralf Braun; Christian G. Huber (pp. 279-288).

The transfer of a gradient method to an isocratic or multistep gradient method employing stationary phase optimized liquid chromatography facilitated a reduction in analysis time by 50% and significantly improved the mass spectrometric detectability of impurities in synthetic thyroid hormones. Four column segments packed with different stationary phases were combined into a single chromatographic column, which allowed the separation and photometric as well as mass spectrometric detection of thyroid compounds in less than 30 min under isocratic- or step gradient elution conditions with 0.10% acetic acid/acetonitrile. Signal instability and baseline drift during detection by negative electrospray ionization time-of-flight mass spectrometry were minimized by optimizing the spray parameters for each individual elution step. This resulted in improved detectabilities and higher mass spectral quality, especially for low-abundance components in the sample mixture. The method was applied to the separation and detection of the low-abundance impurities formed upon the thermal stressing of a sample of synthetic levothyroxine.

Keywords: Liquid chromatography; Electrospray ionization mass spectrometry; Stationary phase optimized liquid chromatography; Thyroxine; Thyroid hormones

Automated normal phase nano high performance liquid chromatography/matrix assisted laser desorption/ionization mass spectrometry for analysis of neutral and acidic glycosphingolipids by Mostafa Zarei; Stephan Kirsch; Johannes Müthing; Laura Bindila; Jasna Peter-Katalinić (pp. 289-297).

The coupling of nano high-performance liquid chromatography (nanoHPLC) with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) via an automatic spotting roboter was developed and adapted for the first time for the analysis of complex mixtures of glycosphingolipids (GSLs). The 2,5-dihydroxybenzoic acid and 6-azo-2-thiothymine matrix systems were adjusted to concurrently meet the requirements for reproducible and homogeneous crystal formation with the liquid chromatography (LC) eluent under the variable LC solvent composition over the course gradient and high ionization efficiency of the GSL species, without the need for recrystallization. Precise adjustment of the automatic spotting parameters in terms of matrix flow rate, on-tip collection time of the matrix/LC eluent solution and the matrix spotting mode, i.e., continuous and discontinuous, was accomplished to collect individually nanoHPLC-separated species within distinct spots and consequently recover by MALDI MS screening all major and minor GSL species in the mixtures. The nanoHPLC/MALDI MS coupling protocol was developed and applied to a mixture of neutral GSLs purified from human erythrocytes and a monosialoganglioside mixture expressed by the murine MDAY-D2 cell line. Additionally, on-line nanoHPLC/MALDI doping with lithium cations of individually separated neutral GSLs was introduced to enhance data interpretation of the GSL MS pattern, while preserving the same level of information and ultimately to enhance structural assignment of components of interest. The method is demonstrated to be highly sensitive, reaching the low femtomole level of detection of individual GSL species and is highlighted as a versatile analytical tool for glycolipidomic studies. Figure Automatic LC/MALDI MS profiling of glycosphingolipids

Keywords: Nano high-performance liquid chromatography; Matrix assisted laser desorption/ionization mass spectrometry; Glycosphingolipids; Automatization

Application of at-line two-dimensional liquid chromatography–mass spectrometry for identification of small hydrophilic angiotensin I-inhibiting peptides in milk hydrolysates by Chris J. van Platerink; Hans-Gerd M. Janssen; Johan Haverkamp (pp. 299-307).

A two-dimensional chromatographic method with mass spectrometric detection has been developed for identification of small, hydrophilic angiotensin I-inhibiting peptides in enzymatically hydrolysed milk proteins. The method involves the further separation of the poorly retained hydrophilic fraction from a standard C₁₈ reversed-phase column on a hydrophilic interaction liquid chromatography (HILIC) column. The latter column is specifically designed for the separation of hydrophilic compounds. Narrow fractions collected from the HILIC column were analysed for their angiotensin I-converting enzyme (ACE) inhibiting potential in an at-line assay. Fractions showing significant inhibition of ACE were analysed by LC–MS for structure elucidation. With this method the main peptides responsible for ACE-inhibition in the hydrophilic part of a milk hydrolysate could be determined. The ACE-inhibiting peptides RP, AP, VK, EK, and EW explained more than 85% of ACE-inhibition by the hydrophilic fraction.

Keywords: Angiotensin-converting enzyme; Blood pressure-lowering peptides; HPLC; MS; Reversed-phase; HILIC

Expanding the scope of MS binding assays to low-affinity markers as exemplified for mGAT1 by Christine Zepperitz; Georg Höfner; Klaus T. Wanner (pp. 309-316).

Following a recently developed concept of MS binding assays based on the quantification of a native marker by LC–MS a procedure to study binding of a low-affinity marker in kinetic, saturation, and competition experiments was established. Separation of bound and unbound marker—the most crucial step of the assay—could be effectively achieved by filtration in a 96-well-format. MS binding assays according to this procedure allowed the reliable characterization of NO 711 binding to mGAT1 in presence of physiological NaCl concentrations. Comparing the results obtained in the present study with those from experiments using 1 mol L⁻¹ NaCl in the incubation milieu reveals remarkable differences with respect to the marker’s affinity and kinetics and to the investigated test compound’s potency. Principle of MS binding assays After incubation of a target with a native marker, bound and unbound marker are separated by filtration. Subsequently, the bound native marker is liberated from the target and finally quantified by LC-MS-MS.

Keywords: MS binding assays; Mass spectrometry; Ligand binding; GAT1; GABA uptake; LC–MS

Using HPTLC/DESI-MS for peptide identification in 1D separations of tryptic protein digests by Sofie P. Pasilis; Vilmos Kertesz; Gary J. Van Berkel; Michael Schulz; Susanne Schorcht (pp. 317-324).

Desorption electrospray ionization mass spectrometry (DESI-MS) was investigated as a method to detect and identify peptides from tryptic digests of cytochrome c and myoglobin separated on ProteoChrom® HPTLC Silica gel 60 F_254s plates and ProteoChrom® HPTLC Cellulose sheets. Full-scan mass spectra and data-dependent tandem mass spectra were acquired in separate plate scans and used to identify peptide ions. Peptide distributions along the development lane were mapped for each separated protein digest. Signal levels ranged over several orders of magnitude. In general, highest signal levels were obtained for the peptides with the highest R _f values on a plate, while peptides with very low R _f values were often not detected. Sequence coverages for cytochrome c were 58% for the digest separated on the silica gel plate and 72% for the separation on the cellulose sheet; myoglobin sequence coverages were 62% and 68% on silica gel and cellulose, respectively. Weak correlations between peptide hydrophilicity and R _f values on the silica gel and cellulose plates were found, with the more hydrophilic peptides having lower R _f values.

Keywords: HPTLC/DESI-MS; Peptides; Mass spectrometry

Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry by Irina Perdivara; Leesa Deterding; Adrian Moise; Kenneth B. Tomer; Michael Przybylski (pp. 325-336).

Using the bottom-up approach and liquid chromatography (LC) in combination with mass spectrometry, the primary structure and sequence microheterogeneity of a plaque-specific anti-β-amyloid (1–17) monoclonal antibody (clone 6E10) was characterized. This study describes the extent of structural information directly attainable by a high-performance LC–tandem mass spectrometric method in combination with both protein database searching and de novo sequence determination. Using trypsin and chymotrypsin for enzymatic digestion, 95% sequence coverage of the light chain and 82% sequence coverage of the heavy chain of the 6E10 antibody were obtained. The primary structure determination of a large number of peptides from the antibody variable regions was obtained through de novo interpretation of the data. In addition, N-terminal truncations of the heavy chain were identified as well as low levels of pyroglutamic acid formation. Surprisingly, pronounced sequence microheterogeneities were determined for the CDR 2 region of the light chain, indicating that changes at the protein level derived from somatic hypermutation of the Ig V_L genes in mature B-cells might contribute to unexpected structural diversity. Furthermore, the major glycoforms at the conserved heavy chain N-glycosylation site, Asn-292, were determined to be core-fucosylated, biantennary, complex-type structures containing zero to two galactose residues. Figure Primary structure and sequence microheterogeneities of a β-amyloid plaque-specific monoclonal antibody were identified by high-performance LC-tandem-mass spectrometry. Sequence heterogeneities of the light chain CDR2 reveal unexpected diversity from VL-gene hypermutations.

Keywords: β-Amyloid antibody; Plaque specific; De novo sequence determination; Microheterogeneity; CDR variations; LC-MS/MS

LC–MS–MS identification of albendazole and flubendazole metabolites formed ex vivo by Haemonchus contortus by Viktor Cvilink; Lenka Skálová; Barbora Szotáková; Jiří Lamka; Risto Kostiainen; Raimo A. Ketola (pp. 337-343).

Resistance of helminth parasites to common anthelminthics is a problem of increasing importance. The full mechanism of resistance development is still not thoroughly elucidated. There is also limited information about helminth enzymes involved in metabolism of anthelminthics. Identification of the metabolites formed by parasitic helminths can serve to specify which enzymes take part in biotransformation of anthelminthics and may participate in resistance development. The aim of our work was to identify the metabolic pathways of the anthelminthic drugs albendazole (ABZ) and flubendazole (FLU) in Haemonchus contortus, a world-wide distributed helminth parasite of ruminants. ABZ and FLU are benzimidazole anthelminthics commonly used in parasitoses treatment. In our ex vivo study one hundred living adults of H. contortus, obtained from the abomasum of an experimentally infected lamb, were incubated in 5 mL RPMI-1640 medium with 10 μmol L⁻¹ benzimidazole drug (10% CO₂, 38 °C) for 24 h. The parasite bodies were then removed from the medium. After homogenization of the parasites, both parasite homogenates and medium from the incubation were separately extracted using solid-phase extraction. The extracts were analyzed by liquid chromatography–mass spectrometry (LC–MS) with electrospray ionization (ESI) in positive-ion mode. The acquired data showed that H. contortus can metabolize ABZ via sulfoxidation and FLU via reduction of a carbonyl group. Albendazole sulfoxide (ABZSO) and reduced flubendazole (FLUR) were the only phase I metabolites detected. Concerning phase II of biotransformation, the formation of glucose conjugates of ABZ, FLU, and FLUR was observed. All metabolites mentioned were found in both parasite homogenates and medium from the incubation.

Keywords: Mass spectrometry; Drug metabolism; Xenobiotics; Glucose conjugation; Biotransformation; Parasitic helminth

Multiresidue analysis of atrazine, diuron and their degradation products in sewage sludge by liquid chromatography tandem mass spectrometry by Aline Ghanem; Philippe Bados; François Perreau; Rachid Benabdallah; Cécile Plagellat; Luiz Felippe de Alencastro; Jacques Einhorn (pp. 345-352).

A multiresidue method has been developed to analyze atrazine (ATZ), diuron (DIU), and their major degradation products, desethylatrazine (DEA), desisopropylatrazine (DIA), and dichlorophenylmethylurea in sewage sludge. Liquid chromatography coupled to electrospray tandem mass spectrometry (LC–ESI-MS–MS) allowed, in the multiple-reaction monitoring mode, the simultaneous analysis of these pesticides in only one run after their extraction with ethyl acetate–dichloromethane 90:10 (v/v) and a cleanup on a Florisil column. Stable isotopically labeled ATZ and DIU were used as internal standards to overcome matrix effects during the pesticide quantification. Using fortified samples, the method gave rise to 86–115% as mean recovery values depending on the analyte. Limits of detection (LODs) and of quantification (LOQs) ranging from 0.3 (DIA) to 1.5 (DEA) μg kg⁻¹ dw and from 0.4 (DIA) to 2.0 (DEA) μg kg⁻¹ dw, respectively, were sufficient to achieve the monitoring of these molecules in sludge from wastewater treatment plants of the Ile-de-France region.

Keywords: Triazine; Phenylurea; Herbicide; Liquid chromatography tandem mass spectrometry; Sewage sludge

Light-scattering and turbidimetric detection of silica colloids in size-exclusion chromatography by Zarina Aspanut; Tomomi Yamada; Lee Wah Lim; Toyohide Takeuchi (pp. 353-359).

Silica colloids were separated by size-exclusion chromatography and monitored by fluorimetric and UV detection. In the former means of detection, silica colloids were visualized by light-scattering. The signal intensity based on the light scattering increased with increasing size of the silica colloids. The maximum intensity was observed at excitation wavelengths around 270–290nm. In UV detection, silica colloids were visualized based on turbidimetry, and the signal intensity also increased with increasing size of the silica colloids and with decreasing detection wavelength. The signal intensities for both light-scattering and turbidimetric detection were a linear function of the concentration of the silica colloids. The detection limit at S/N = 3 for 78-nm colloids was 0.06 ppm for light-scattering detection whereas the LOD was 2.3 ppm for UV detection. Effects of mobile phase conditions and flow rate on resolution and peak shape were examined. Use of phosphate buffer allowed the separation of silica colloids of different sizes in size-exclusion chromatography.

Keywords: Silica colloids; Light-scattering detection; Turbidimetric detection; Size-exclusion chromatography

Establishment of “one-piece” large-gel 2-DE for high-resolution analysis of small amounts of sample using difference gel electrophoresis saturation labelling by B. Sitek; B. Sipos; K. Pfeiffer; M. Grzendowski; G. Poschmann; E. Hawranke; K. Köper; G. Klöppel; H. E. Meyer; K. Stühler (pp. 361-365).

Large-gel two-dimensional gel electrophoresis (2-DE) is the method of choice for high-resolution proteome analysis of complex protein mixtures. Until now, however, the advantages of large 2-DE in combination with multiplexed fluorescence dye protein labelling has been complicated by the separate handling and analysis of the second-dimension gels. Therefore, we adapted the large 2-DE procedure allowing us to run “one-piece” large 2-DE gels (40 cm × 30 cm) in the second dimension for high resolution proteome analysis. Here, we show that in combination with fluorescence dye protein saturation labelling “one-piece” large 2-DE enables analysis of small amounts of sample (3 µg protein) for high-resolution proteome analysis.

Keywords: Differential proteome analysis; Saturation DIGE; Fluorescence dye; Microdissection; Large two-dimensional electrophoresis (2-DE); Biomarker

RP-HPLC determination of the lipophilicity of bispyridinium reactivators of acetylcholinesterase bearing a but-2-ene connecting linker by Kamil Musilek; Josef Jampilek; Jiri Dohnal; Daniel Jun; Frank Gunn-Moore; Martin Dolezal; Kamil Kuca (pp. 367-372).

New acetylcholinesterase reactivators with either a (E) or (Z)-but-2-ene connecting linker were recently prepared. The purity of the compounds was checked by HPLC and was found to be sufficient for in-vitro screening. All the discussed bispyridinium reactivators were analyzed by reversed-phase high performance liquid chromatography (RP-HPLC) to measure lipophilicity. The procedure was performed under isocratic conditions with methanol as organic modifier in the mobile phase using an end-capped non-polar C₁₈ stationary phase RP column. Relationships between the lipophilicity (logarithm of the RP-HPLC capacity factor, log k) and chemical structures of the studied compounds are discussed. Lipophilicity was different for the (E) and (Z) compounds and varied among the compounds in each of these groups. The lipophilicity differences also indicated an apparent influence of intramolecular interactions. Lipophilicity calculations (log P/Clog P) by means of commonly used software were not successful due to the presence of quaternary nitrogen atoms in the molecules of the reactivators.

Keywords: Acetylcholinesterase; Reactivator; RP-HPLC; Purity; Lipophilicity

Comprehensive two-dimensional liquid chromatography in the analysis of antioxidant phenolic compounds in wines and juices by Maarit Kivilompolo; Vít Obůrka; Tuulia Hyötyläinen (pp. 373-380).

A novel comprehensive two-dimensional liquid chromatographic (LC×LC) system was developed for the quantification of antioxidant phenolic compounds in wine and juice. The system allows parts of the sample that are well separated in the first column to pass directly to the detector after the first column, while the rest of the sample proceeds to the second column. The optimised LC×LC system employed a combination of two C18 columns, the latter column with an ion-pair reagent (tetrapentylammonium bromide). The relative standard deviations (RSD) for the retention times were better than 0.01% in the first dimension and on average 6.3% in the second. The RSD values of the peak volumes varied from 3% (protocatechuic acid) to 13% (caffeic acid, n = 3, 10 μg/ml).

Keywords: Comprehensive two-dimensional liquid chromatography; Wine; Phenolic compounds

Identification of changes in Triticum durum L. leaf proteome in response to salt stress by two-dimensional electrophoresis and MALDI-TOF mass spectrometry by Giuseppe Caruso; Chiara Cavaliere; Chiara Guarino; Riccardo Gubbiotti; Patrizia Foglia; Aldo Laganà (pp. 381-390).

In order to understand the molecular basis of salt stress response, a proteomic approach, employing two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), was used to identify proteins affected by salinity in wheat (Triticum durum ‘Ofanto’). Identification of proteins, whose levels were altered, was performed by comparing protein patterns of salt-treated and control plants. A set of control plants was grown without NaCl addition under the same conditions as the salt-treated plants. Proteins were extracted from the leaves of untreated and NaCl-treated plants, and resolved using 24-cm immobilized pH gradient strips with a pH 4–7 linear gradient in the first dimension and a 12.5% sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension; the gels were stained with Coomassie and image analysis was performed. Quantitative evaluation, statistical analyses and MALDI-TOF MS characterization of the resolved spots in treated and untreated samples enabled us to identify 38 proteins whose levels were altered in response to salt stress. In particular, ten proteins were downregulated and 28 were upregulated. A possible role of these proteins in response to salinity is discussed.

Keywords: Matrix-assisted laser desorption/ionization time of flight; Proteomics; Salt stress; Triticum durum ; Wheat; Two-dimensional electrophoresis

Mechanism of Coomassie brilliant blue G-250 binding to proteins: a hydrophobic assay for nanogram quantities of proteins by Christos D. Georgiou; Konstantinos Grintzalis; George Zervoudakis; Ioannis Papapostolou (pp. 391-403).

We investigated the mechanism of Coomassie brilliant blue G-250 (CBB) binding to proteins in order to develop a protein assay with the maximum possible sensitivity. We found that the neutral ionic species of CBB binds to proteins by a combination of hydrophobic interactions and heteropolar bonding with basic amino acids. On the basis of these findings, we developed a very sensitive hydrophobic assay for proteins (at the nanogram level) using the hydrophobic reagents ammonium sulfate and trichloroacetic acid under pH conditions that increase neutral species concentration in the assay reagent in order to enhance the binding of more CBB dye molecules per protein molecule than in previous CBB-based assays.

Keywords: Coomassie brilliant blue G-250; Proteins; Quantification

Microarray-based detection of Korean-specific BRCA1 mutations by Cheulhee Jung; Seong-Chun Yim; Dae-Yeon Cho; Ho Nam Chang; Hyun Gyu Park (pp. 405-413).

A reliable multiplex assay procedure to detect human genetic mutations in the breast cancer susceptibility gene BRCA1 using zip-code microarrays and single base extension (SBE) reactions is described. Multiplex PCR amplification was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in subsequent multiplex SBE reactions using bifunctional primers carrying a unique complementary zip sequence in addition to a mutation-site-specific sequence. The SBE primers, terminating one base before their mutation sites, were extended by a single base at a mutation site with a corresponding biotin-labeled ddNTP. Hybridization of the SBE products to zip-code microarrays was followed by staining with streptavidin–Cy3, leading to successful genotyping of several selected BRCA1 mutation sites with wild-type and heterozygote mutant samples from breast cancer patients. This work has led to the development of a reliable DNA microarray-based system for the diagnosis of human genetic mutations.

Keywords: DNA chip; BRCA; Mutation detection; Zip-code microarray; Single base extension

Antioxidative effect of Bacteroides thetaiotaomicron extracts: superoxide dismutase identification by Anne-Cécile Hochart-Behra; Josette Behra-Miellet; Julie Sam; Hervé Drobecq; Bernard Gressier; Michel Luyckx; Thierry Dine; Claude Brunet; Luc Dubreuil (pp. 415-423).

Bacteroides thetaiotaomicron, a bowel anaerobic commensal, seems to release enzymes detoxifying reactive oxygen species according to our recent work. This opportunistic pathogen would be beneficial in the case of an inflammatory process. To explore its role after an oxidative or nutritive stress, six to seven separate experiments were performed. The bacteria were grown on media restricted in growth factors or supplemented with bile. Their viability was checked after surface protein extraction. The extracts underwent 2D electrophoresis. Gel images were statistically analysed to construct “master” gels. Proteins were identified (peptide-mass fingerprinting technique). The effect of each extract on superoxide anions was evaluated (spectrophotometric method). Superoxide dismutase was identified and a major superoxide anion inhibition was shown by extracts obtained after a nutritive and oxidative stress without significant bacterial death. So, a therapeutic antioxidant potential is firmly hoped for. Figure These intestinal commensal Gram negative anaerobic bacilli are able to release a high level of functional superoxide dismutase when grown in minimal medium.

Keywords: Bioassays; Bioanalytical methods; Enzymes; Pharmaceuticals; Proteomics

Application of SPME to the determination of alkylphenols and bisphenol A in cyanobacteria culture media by Teodor Stoichev; Mafalda S. Baptista; M. Clara P. Basto; Pedro N. Carvalho; M. Teresa S. D. Vasconcelos (pp. 425-432).

In order to survey the influence of estrogenic compounds on cyanobacteria, solid-phase microextraction (SPME) with a carbowax-divinylbenzene fibre was used for the determination of tert-octylphenol (tert-OP), n-nonylphenol (n-NP), technical nonylphenol (tech-NP) and bisphenol A (BPA) in cyanobacteria culture media by gas chromatography with flame ionization detection. Determinations were carried out without derivatization in deionized water and filtered culture media. A comparison between f2 and Fraquil culture media was performed, which showed that only f2 allowed quantitative recoveries. Headspace SPME with salting out, requiring only 10 mL of sample, was suitable for tert-OP, n-NP, and tech-NP determination with limits of detection (LOD) of <0.05 μg L⁻¹. For BPA, direct immersion SPME could provide a LOD of 1 μg L⁻¹. Automated sampling allowed reproducible extraction. No exudate substances overlapped with the studied compounds during the chromatographic separation and no matrix effects were observed. Ecotoxicity tests can be performed by single spiking of tert-OP and tech-NP and multiple spiking of n-NP due to its lower stability.

Keywords: Nonylphenol; Octylphenol; Bisphenol A; Gas chromatography; Solid-phase microextraction; Phytoplankton

Noninvasive and nondestructive NMR, Raman and XRF analysis of a Blaeu coloured map from the seventeenth century by K. Castro; S. Pessanha; N. Proietti; E. Princi; D. Capitani; M. L. Carvalho; J. M. Madariaga (pp. 433-441).

A complete multianalytical study of a hand-coloured map from the seventeenth century is presented. The pigments atacamite, massicot, minium, gypsum, carbon black and vermilion were determined by means of XRF and Raman spectroscopy. The state of conservation of the cellulosic support was monitored by means of unilateral NMR. The analysis was nondestructive and noninvasive, and thus several spectra were collected from the same areas, yielding more reliable results without damaging the artwork. The role of copper pigments in the oxidation processes observed in the cellulosic support is discussed, as well as the possible provenance of atacamite as a raw material instead of as a degradation product of malachite.

Keywords: Raman; FTIR; NMR; Pigment; XRF; Cellulose; Degradation

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Analytical and Bioanalytical Chemistry (v.391, #1)