Analytical and Bioanalytical Chemistry (v.394, #6)

Career break, not career broken by Sara Shinton (1509-1511).
started her professional life as a chemist, following her first degree in Chemical and Analytical Science with a PhD in Physical Chemistry. After a brief period in post-doctoral research, she discovered an interest in transferable skills whilst teaching undergraduate chemists communication skills. After retraining as a university careers adviser, in 2000 she founded a company [ www.shintonconsulting.com ] and now delivers careers guidance and skills training to academic researchers and scientists across the UK and Europe.

Focus on analytical science in Spain (JAI 2008) by Joan O. Grimalt; Alfredo Sanz-Medel (1515-1516).
is Research Professor and Director of the Institute of Environmental Assessment and Water Research (IDÆA) of the Spanish Council for Scientific Research (CSIC). His professional activity has developed within environmental organic geochemistry. More specifically, his research is devoted to the study of the natural and anthropogenic organic compounds as markers of the health status of ecosystems and living organisms (including humans). He has published about 460 scientific papers, most of them in international peer-reviewed journals. Presently, he is President of the Spanish Society of Chromatography and Related Techniques. He has received the King Jaume I award devoted to the preservation of environment (Valencian Autonomous Government, 2005), the Environment Award of the Catalan Academia (2001), and the Award of Scientific Research of the City of Barcelona (2000). has been Professor in the Department of Physical and Analytical Chemistry of Oviedo University (Spain) since 1982. He is author or coauthor of around 500 scientific publications in international journals, several patents, and books. His present research interests include new atomic detectors and ion sources for ultratrace analysis using plasmas, new molecular optical sensors, particularly those based on the use of quantum dots, as well as hybrid techniques, coupling a separation unit and an atomic detector, for ultratrace and trace metal speciation to solve biological and environmental problems and speciation for proteomics, integrating mass spectrometry (MS) “molecular” (matrix-assisted laser desorption/ionization and electrospray MS n ) and “atomic” [inductively coupled plasma (ICP) MS] techniques and introducing the extensive use of ICP-MS to carry out “heteroatom-tagged proteomics,” both for qualitative and for quantitative purposes. He has been an editor of Analytical and Bioanalytical Chemistry since 2002. At Euroanalysis 2007 in Antwerp he received the Robert Kellner Award.

Plasma fingerprinting with GC-MS in acute coronary syndrome by M. Vallejo; A. García; J. Tuñón; D. García-Martínez; S. Angulo; J.L. Martin-Ventura; L. M. Blanco-Colio; P. Almeida; J. Egido; C. Barbas (1517-1524).
New biomarkers of cardiovascular disease are needed to augment the information obtained from traditional indicators and to illuminate disease mechanisms. One of the approaches used in metabolomics/metabonomics for that purpose is metabolic fingerprinting aiming to profile large numbers of chemically diverse metabolites in an essentially nonselective way. In this study, gas chromatography-mass spectrometry was employed to evaluate the major metabolic changes in low molecular weight plasma metabolites of patients with acute coronary syndrome (n = 9) and with stable atherosclerosis (n = 10) vs healthy subjects without significant differences in age and sex (n = 10). Reproducible differences between cases and controls were obtained with pattern recognition techniques, and metabolites accounting for higher weight in the classification have been identified through their mass spectra. On this basis, it seems inherently plausible that even a simple metabolite profile might be able to offer improved clinical diagnosis and prognosis, but in addition, specific markers are being identified.
Keywords: Acute coronary syndrome; Atherosclerosis; Metabonomics; Metabolomics; Pattern recognition; Separation techniques; Stroke

Determination of alkylphenols and alkylphenol ethoxylates in sewage sludge: effect of sample pre-treatment by María Fernández-Sanjuan; Anna Rigol; Angels Sahuquillo; Sonia Rodríguez-Cruz; Silvia Lacorte (1525-1533).
A complete characterization of sewage sludge collected from five biological waste water treatment plants was done to determine physico-chemical parameters, heavy metals and alkylphenols, making special emphasis on sampling, homogenization, and sample pre-treatment. Ultrasonic extraction followed by gas chromatrography coupled with mass spectrometry was used to evaluate the effect of sample pre-treatment (untreated sample, freeze-drying, drying at 40 °C or drying at 100 °C) on the concentration of octylphenol (OP), nonylphenol (NP) and nonylphenol ethoxylates (NP1EO, NP2EO). Untreated samples and samples dried at 100 ºC gave concentration levels up to 62% and 89% lower, respectively, than freeze-dried samples. In 50% of cases, freeze-dried samples led to significantly higher concentrations than those obtained by drying at 40 °C. Thus, freeze-drying is the recommended sample pre-treatment to prevent possible losses of OP, NP, and NP1EO. Using this methodology, concentrations detected were from 3.2 to 199 mg kg−1 being NP followed by NP1EO found in highest concentration. The total concentration of NP and NP1EO exceeded the limit of 50 mg kg−1 proposed by the draft European directive on sewage sludge in three out of five samples studied. Contrarily, heavy metals were below the legislated values.
Keywords: Alkylphenols; Heavy metals; Gas chromatography–mass spectrometry; Sewage sludge; Drying

Determination of metalaxyl and identification of adjuvants in wettable powder pesticide technical formulas by Eva Pose-Juan; Raquel Rial-Otero; Elena Martínez-Carballo; Eugenio López-Periago; Jesús Simal-Gándara (1535-1544).
Foliar runoff is one of the most important processes affecting off-target movement of fungicides. In this way, Ridomil® Gold Plus and Ridomil® Gold MZ are two types of wettable powder technical formulations which contain metalaxyl and they are used for such a purpose. A method for quantitative determination of metalaxyl in pesticide formulas has been developed, validated, and subsequently applied to Ridomil® Gold Plus and Ridomil® Gold MZ. The method employs liquid–liquid extraction followed by liquid chromatography coupled with UV detection (LC–UV), using gas chromatography coupled with mass spectrometry as confirmation technique and to carry out a screening of organic adjuvants of these two selected pesticide formulas. Metalaxyl of 26.5 and 41 g/kg was detected in Ridomil® Gold Plus and Ridomil® Gold MZ, close to the manufacture specified level of 25 and 40 g/kg, respectively. Activator and utility adjuvants were detected in these two wettable powder technical formulations. Only methyl-ester-based surfactants were found within the group of nonionic surfactants, but the long-term fates of most adjuvants in soils and elsewhere in the environment are largely unknown, partially because of the lack of long-term monitoring data.
Keywords: Formulation; Ridomil® Gold Plus and Ridomil® Gold MZ; Metalaxyl; Adjuvants; Pesticides/endocrine disruptors; HPLC; Surfactants

Epicatechin, procyanidins, and phenolic microbial metabolites after cocoa intake in humans and rats by Mireia Urpi-Sarda; Maria Monagas; Nasiruddin Khan; Rosa M. Lamuela-Raventos; Celestino Santos-Buelga; Emilio Sacanella; Margarida Castell; Joan Permanyer; Cristina Andres-Lacueva (1545-1556).
Proanthocyanidins, flavonoids exhibiting cardiovascular protection, constitute a major fraction of the flavonoid ingested in the human diet. Although they are poorly absorbed, they are metabolized by the intestinal microbiota into various phenolic acids. An analytical method, based on an optimized 96-well plate solid-phase extraction (SPE) procedure and liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) for the analysis of 19 phenolic microbial metabolites and monomeric and dimeric flavanols in urine samples, was developed and validated. Human urine samples were obtained before and after ingestion of an acute consumption of 40 g of soluble cocoa powder and rat urines before and after the prolonged administration (2 weeks) of different diets composed of natural cocoa powder. The mean recovery of analytes using the new SPE-LC-MS/MS method ranged from 87% to 109%. Accuracy ranged from 87.5% to 113.8%, and precision met acceptance criteria (<15% relative standard deviation). Procyanidin B2 has been detected and quantified for the first time in human and rat urine after cocoa consumption. Changes in human and rat urinary levels of microbial phenolic acids and flavanols were in the range of 0.001–59.43 nmol/mg creatinine and of 0.004–181.56 nmol/mg creatinine, respectively. Major advantages of the method developed include reduction of laboratory work in the sample preparation step by the use of 96-well SPE plates and the sensitive measurement of a large number of metabolites in a very short run time, which makes it ideal for use in epidemiological studies.
Keywords: Cocoa; Epicatechin; Procyanidin; Microbial metabolites; LC-MS/MS

A sensitive and simple analytical method has been developed for determination of Sb(III), Sb(V), Se(IV), Se(VI), Te(IV), Te(VI), and Bi(III) in garlic samples by using hydride-generation–atomic-fluorescence spectrometry (HG–AFS). The method is based on a single extraction of the inorganic species by sonication at room temperature with 1 mol L−1 H2SO4 and washing of the solid phase with 0.1% (w/v) EDTA, followed by measurement of the corresponding hydrides generated under two different experimental conditions directly and after a pre-reduction step. The limit of detection of the method was 0.7 ng g−1 for Sb(III), 1.0 ng g−1 for Sb(V), 1.3 ng g−1 for Se(IV), 1.0 ng g−1 for Se(VI), 1.1 ng g−1 for Te(IV), 0.5 ng g−1 for Te(VI), and 0.9 ng g−1 for Bi(III), in all cases expressed in terms of sample dry weight.
Keywords: Antimony; Selenium; Tellurium; Bismuth; Inorganic species; Garlic; HG–AFS

Application of a trazodone-selective electrode to pharmaceutical quality control and urine analyses by M. Soledad García; Joaquín Ortuño; M. Isabel Albero; María Cuartero (1563-1567).
A trazodone-selective electrode for application in pharmaceutical quality control and urine analysis was developed. The electrode is based on incorporation of a trazodone-tetraphenylborate ion exchanger in a poly(vinyl chloride) membrane. The electrode showed a fast, stable and Nernstian response over a wide trazodone concentration range (5 × 10−5−1 × 10−2 M) with a mean slope of 59.3 ± 0.9 mV/dec of concentration, a mean detection limit of 1.8 × 10−5 ± 2.2 × 10−6 M, a wide working pH range (5–7.5) and a fast response time (less than 20 s). The electrode also showed good accuracy, repeatability, reproducibility and selectivity with respect to some inorganic and organic compounds, including the main trazodone metabolite. The electrode provided good analytical results in the determination of trazodone in pharmaceuticals and spiked urine samples; no extraction steps were necessary. Dissolution testing of trazodone tablets, in different conditions of pH and particle size, based on a direct potentiometric determination with the new selective electrode is presented as well.
Keywords: Ion-selective electrode; Trazodone determination; Pharmaceuticals; Urine samples; Dissolution testing

Halogenated molecularly imprinted polymers for selective determination of carbaryl by phosphorescence measurements by Adrián Alvarez-Diaz; José M. Costa; Rosario Pereiro; Alfredo Sanz-Medel (1569-1576).
A highly selective molecularly imprinted polymer (MIP) for the recognition of the pesticide carbaryl in water has been synthesized using halogenated bisphenol A compounds as one of the polymeric precursors and carbaryl as the template molecule. On the basis of the heavy-atom effect, both the brominated and the iodinated MIPs allowed analyte detection by room-temperature-phosphorescence measurements. In the presence of an oxygen scavenger (sodium sulphite) the halide, included in the polymeric structure, induced efficient room-temperature phosphorescence of the analyte (once it had been selectively retained by the MIP). The MIP cavity can be easily regenerated for subsequent sample injections with 2 mL methanol. The optosensing system developed has demonstrated high selectivity for carbaryl, even in the presence of other luminophores that could be unspecifically adsorbed onto the MIP surface. Under optimal experimental conditions, the detection limit for the target molecule was 4 µg/L (3-mL sample injection volume), and the linear range extended up to 1 mg/L of the analyte. Good reproducibility was achieved (a relative standard deviation of 3% was obtained for ten replicates of 150 µg/L carbaryl). The synthesized sensing material showed good stability for at least 3 months after preparation. Finally, the applicability to carbaryl determination in real samples was evaluated through the successful determination of the pesticide in spiked mineral and tap water samples. Figure Schematic diagram of carbaryl recognition process by an halogenated molecularly imprinted polymer for room temperature phosphorescence detection of the analyte.
Keywords: Carbaryl; Molecular imprinting; Room-temperature phosphorescence; Biomimetic optosensing; Water analysis

In this work, the hyphenation of the multisyringe flow injection analysis technique with a 100-cm-long pathlength liquid core waveguide has been accomplished. The Cl/Hg(SCN)2/Fe3+ reaction system for the spectrophotometric determination of chloride (Cl) in waters was used as chemical model. As a result, this classic analytical methodology has been improved, minimizing dramatically the consumption of reagents, in particular, that of the highly biotoxic chemical Hg(SCN)2. The proposed method features a linear dynamic range composed of two steps between (1) 0.2–2 and (2) 2–8 mg Cl L−1, thus extended applicability due to on-line sample dilution (up to 400 mg Cl L−1). It also presents improved limits of detection and quantification of 0.06 and 0.20 mg Cl L−1, respectively. The coefficient of variation and the injection throughput were 1.3% (n = 10, 2 mg Cl L−1) and 21 h−1. Furthermore, a very low consumption of reagents per Cl determination of 0.2 μg Hg(II) and 28 μg Fe3+ has been achieved. The method was successfully applied to the determination of Cl in different types of water samples. Finally, the proposed system is critically compared from a green analytical chemistry point of view against other flow systems for the same purpose.
Keywords: Green analytical chemistry; Multisyringe flow injection analysis; Liquid core waveguide; Long pathlength spectrophotometry; Chloride determination; Mercury thiocyanate

A method for the simultaneous determination of seven commonly used artificial sweeteners in water is presented. The analytes were extracted by solid phase extraction using Bakerbond SDB 1 cartridges at pH 3 and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry in negative ionization mode. Ionization was enhanced by post-column addition of the alkaline modifier Tris(hydroxymethyl)amino methane. Except for aspartame and neohesperidin dihydrochalcone, recoveries were higher than 75% in potable water with comparable results for surface water. Matrix effects due to reduced extraction yields in undiluted waste water were negligible for aspartame and neotame but considerable for the other compounds. The widespread distribution of acesulfame, saccharin, cyclamate, and sucralose in the aquatic environment could be proven. Concentrations in two influents of German sewage treatment plants (STPs) were up to 190 μg/L for cyclamate, about 40 μg/L for acesulfame and saccharin, and less than 1 μg/L for sucralose. Removal in the STPs was limited for acesulfame and sucralose and >94% for saccharin and cyclamate. The persistence of some artificial sweeteners during soil aquifer treatment was demonstrated and confirmed their environmental relevance. The use of sucralose and acesulfame as tracers for anthropogenic contamination is conceivable. In German surface waters, acesulfame was the predominant artificial sweetener with concentrations exceeding 2 μg/L. Other sweeteners were detected up to several hundred nanograms per liter in the order saccharin ≈ cyclamate > sucralose. Figure Some artificial sweeteners are excreted unchanged and in particular acesulfame is a perfect tracer for municipal waste water
Keywords: Artificial sweeteners; Sucralose; Acesulfame; Surface water; Waste water; Soil aquifer treatment

Study of uptake and loss of silica nanoparticles in living human lung epithelial cells at single cell level by Isaac Stayton; Jeffrey Winiarz; Katie Shannon; Yinfa Ma (1595-1608).
The toxicology of nanomaterials is a blooming field of study, yet it is difficult to keep pace with the innovations in new materials and material applications. Those applications are quickly being introduced in research, industrial, and consumer settings. Even though the cytotoxicity of many types of nanoparticles has been demonstrated, the behavior of those particles in a biological environment is not yet fully known. This work characterized the following over time: protein adsorption on silica particle surfaces, the internalization of particles in human lung carcinoma (A549) cells when coated with different specific proteins or no proteins at all, and the cellular loss of particles following the removal of extracellular particles. Proteins were shown to quickly saturate the particle surface, followed by a competitive process of particle agglomeration and protein adsorption. Uptake of particles peaked at 8–10 h, and it was determined that, in this system, the charge of the protein-coated particles changed the rate of uptake if the charge difference was great enough. Cells internalized particles lacking any adsorbed proteins with approximately 3 times the rate of protein-coated particles with the same charge. Although particles exited cells over time, the process was slower than uptake and did not near completion within 24 h. Finally, analysis at the single cell level afforded observations of particle agglomerates loosely associated with cell membranes when serum was present in the culture medium, but in the absence of serum, particles adhered to the dish floor and formed smaller agglomerates on cell surfaces. Although data trends were easily distinguished, all samples showed considerable variation from cell to cell. Figure Silica-capped fluorescent semiconductor nanoparticles as internalized by human lung epithelial cells and adsorbed to a glass substrate in protein-free culture medium.
Keywords: Human lung epithelial cells; Silica nanoparticles; Cell uptake; Single cell imaging

Cigarette smoke extract induced protein phosphorylation changes in human microvascular endothelial cells in vitro by Jeffery S. Edmiston; Jason W. Flora; Mariano J. Scian; Guoya Li; Gaurav S. J. B. Rana; Timothy B. Langston; Tapas K. Sengupta; Willie J. McKinney (1609-1620).
Phosphorylation is the most widely studied posttranslational modification (PTM) and is an important regulatory mechanism used during cellular responses to external stimuli. The kinases and phosphatases that regulate protein phosphorylation are known to be affected in many human diseases. Cigarette smoking causes cardiovascular disease (CVD). Endothelial cells play a pivotal role in CVD initiation and development; however, there have been limited investigations of the specific signaling cascades and protein phosphorylations activated by cigarette smoke in endothelial cells. The purpose of this research was to better understand the differential protein phosphorylation in endothelial cells stimulated with extracts of cigarette smoke total particulate matter (CS-TPM) in vitro. Human microvascular endothelial cells were exposed in vitro to CS-TPM at concentrations that were shown to cause endothelial cell dysfunction. The phosphorylated proteins were isolated using phosphoprotein-specific chromatography, followed by enzymatic digestion and nano-flow capillary liquid chromatography (ncap-LC) coupled to high resolution mass spectrometry. This study putatively identified 94 proteins in human microvascular endothelial cells that were differentially bound to a phosphoprotein-specific chromatography column following exposure to CS-TPM suggesting differential phosphorylation. Pathway analysis has also been conducted and confirmations of several observations have been made using immunoaffinity-based techniques (e.g., Western blotting).
Keywords: Bioanalytical methods; Amino acids; Peptides; Cell systems; Single cell analysis; Genomics; Proteomics; HPLC

The kinetics of simulated low-energy daylight (UVA–vis) and powerful combined ultraviolet B and A (UVB–UVA) induced direct and indirect phototransformations of four pharmaceuticals, i.e., ibuprofen, metoprolol, carbamazepine, and warfarin, which were investigated in dilute solutions of pure laboratory and natural humic waters. The results strengthen the essential function of natural chromophores in dissolved organic material (CDOM) as principal photosensitizer toward indirect phototransformations of pharmaceuticals in natural conditions under available low-energy UVA–vis and slight UVB radiations. The results confirmed that organic micropollutants are able to undergo a direct photolysis if their absorbance spectra overlap the spectral range of the available radiation but only if the radiation is strong enough, e.g., ibuprofen is able to undergo only indirect photolysis via different pathways in all realistic conditions. The action of nitrate anions as photosensitizers in the applied conditions proved to be of little importance. High-performance size-exclusion chromatographic experiments verified that the rate constants obtained under the low-energy UVA–vis and powerful UVB–UVA irradiations for the decreased amounts of the two largest molecular size fractions of CDOM were quite close to the rate constants detected for the increased amounts of the next five molecular size fractions with smaller molecular sizes. The decreased contents of the two largest molecular size fractions correlated quite well with the decreased contents of the studied pharmaceuticals under the low-energy UVA–vis irradiation process but somewhat less under the powerful UVB–UVA irradiation. The photochemically induced decomposition of the CDOM aggregates appears to increase the amounts of smaller molecular size fractions and simultaneously produce via CDOM-stimulated radical reactions indirect structural transformations of pharmaceuticals. Apparent quantum yields were estimated for the transformation–degradation of the two largest molecular-size CDOM aggregates under low-energy UVA–vis and powerful UVB–UVA irradiations. Figure Structural difference between CDOM and pharmaceuticals studies
Keywords: Pharmaceuticals; Phototransformation; Sensitizers; Natural humic water; Molecular mixtures; Molecular sizes

Locked nucleic acid (LNA) is a deoxyribonucleotide analogue with an unusual ‘locked’ furanose conformation. LNA-modified oligonucleotide probes have demonstrated an enhanced binding affinity towards their complementary strands; however, their potential to discriminate non-complementary hybridization of mismatches has not been explored. In this study, we investigated the effect of the chemical nature of LNA nucleobases on the hybridization stability and the capability of LNA-modified oligonucleotides to discriminate the LNA:DNA mismatched base pairs. It was observed that LNA modification indeed improves the discrimination capability of oligonucleotides by increasing the melting temperature differences between the complementary duplexes and hybrids containing mismatches. Particularly, LNA purines offer a greater potential to recognize the mismatches than LNA pyrimidines and DNA purines. Real-time PCR experiments further confirmed that LNA modifications at the 3′-end are more effective. The results and conclusions in this study provide useful information for hybridization-based nucleic acid analysis where designing sound oligonucleotide probes is crucial to the success of the analyses.
Keywords: Locked nucleic acid; Hybridization; Discrimination; Mismatch

Multicomponent analyses of chiral samples by use of regression analysis of UV–visible spectra of cyclodextrin guest–host complexes by Sayo O. Fakayode; Pamlea N. Brady; David A. Pollard; Abdul K. Mohammed; Isiah M. Warner (1645-1653).
We report the first combined use of analytical spectroscopy, guest–host chemistry, and multivariate regression analysis for determination of enantiometric composition of multicomponent samples of chiral analytes. Sample solutions containing multicomponent analytes of ephedrine, tryptophan, propranolol, and proline of varying enantiomeric composition with beta-cyclodextrin (BCD) or methyl-beta-cyclodextrin (Me-BCD) as chiral host molecules were investigated using ultraviolet (UV)–visible spectroscopy. The interactions of enantiomers of chiral analytes with chiral hosts resulted in the formation of transient diastereomeric inclusion complexes with varying spectral properties. Multivariate analysis using partial-least-square (PLS) regression was used to correlate subtle changes in the UV–visible spectra of the guest–host complexes with the enantiomeric composition of the calibration samples. These PLS regressions were carefully optimized and then used to predict the enantiomeric composition of multicomponent chiral analytes of validation samples. The results of these validation studies demonstrate the predictive ability of the regression models for determination of future enantiomeric composition of samples. The accuracy of the models to correctly predict the enantiomeric composition of samples, evaluated by use of the root mean square percent relative error (RMS%RE) was analyte and chiral host dependent. In general, better prediction of enantiomeric composition of samples and low RMS%RE values were obtained when Me-BCD was used as the chiral host. The analyses procedure reported here is simple, rapid, and inexpensive. In addition, this approach does not require prior separation of chiral analytes, thus reducing analysis time and eliminating the need for expensive chiral columns.
Keywords: UV–visible spectroscopy; Guest–host complexes; Partial-least-square regression; Multicomponent chiral analytes; Chiral analysis

Pattern recognition analysis for 1H NMR spectra of plasma from hemodialysis patients by Masako Fujiwara; Takeshi Kobayashi; Takahiro Jomori; Yutaka Maruyama; Yoshitomo Oka; Hiroshi Sekino; Yutaka Imai; Kazuhisa Takeuchi (1655-1660).
1H NMR spectroscopic and pattern recognition-based methods (NMR-PR) were applied to the metabolic profiling studies on hemodialysis (HD). Plasma samples were collected from 37 patients before and after HD and measured by 600 MHz NMR spectroscopy. Each spectrum was data-processed and subjected to principal component analysis for pattern recognition. Spectral patterns of plasma between pre- and post-dialyses were clearly discriminated, together with significant fluctuations in the levels of creatinine, trimethylamine-N-oxide, glucose, lactate, and acetate, which were quantitated. We have first observed the significant elevation of lactate levels in post-dialysis plasma. The present study has demonstrated the high feasibility of NMR-PR method for monitoring the dialysis condition and comprehensive profiling of the change of low-molecular-weight metabolites in HD. Figure PCA for 1H NMR spectra of plasma from HD patients
Keywords: Lactate; Acetate; TMAO; PCA; Renal failure; Metabolomics

Identification and quantification of glucosinolates in rapeseed using liquid chromatography–ion trap mass spectrometry by Silvia Millán; M. Carmen Sampedro; Patricia Gallejones; Ander Castellón; Maria L. Ibargoitia; M. Aranzazu Goicolea; Ramón J. Barrio (1661-1669).
A rapid and sensitive method for the speciation and quantification of glucosinolates in rapeseed is described. The method combines liquid chromatography (LC) with ion trap mass spectrometry (ITMS) detection. Electrospray ionization (ESI) has been chosen as the ionization technique for the on-line coupling of LC with ITMS. Glucosinolates are extracted from different rapeseeds with MeOH and the extracts are cleaned-up by solid phase extraction with Florisil cartridges. Aqueous extracts are injected into LC system coupled to an ITMS, leading to accurately quantify eight of the most important glucosinolates in rapeseed, by MS2 mode and confirming their structure by MS3 acquisition. All the glucosinolates found in rapeseeds provide good signals corresponding to the deprotonated precursor ion [M-H]. The method is reliable and reproducible, and detection limits range from 0.5 nmol g−1 to 3.7 nmol g−1 when 200 mg of dried seeds of certified reference material are analyzed. Within-day and between-day RSD percentages range between 2.4–14.1% and 3.9–16.9%, respectively. The LC-ESI-ITMS-MS method described here allows for a rapid assessment of these metabolites in rapeseed without a desulfatation step. The overall process has been successfully applied to identify and quantify glucosinolates in rapeseed samples.
Keywords: Oily seeds; Glucosinolates; Liquid chromatography; Ion trap mass spectrometry

This work describes the use of a new dedicated laboratory-made micro X-ray diffraction system for detecting the phases present in cross-sections of artworks. As an example, the phases present in samples from gilding ceramics and stone sculptures from the heritage of Seville (Spain) were successfully detected using this new system, which takes advantage of various devices developed for synchrotron radiation, and is complemented by the information provided by other techniques.
Keywords: Dedicated laboratory-made micro X-ray-diffraction system; Cross-sections; Spanish cultural heritage; Gilding

A fluorescent assay for the evaluation of inhibitors of fatty acid amide hydrolase (FAAH) is described. Microsomes from rat brain served as enzyme source. N-(2-Hydroxyethyl)-4-pyren-1-ylbutanamide was designed and synthesized as novel fluorogenic substrate. For substrate solubilization, Triton X-100 was employed. The FAAH activity was determined directly without further sample clean-up by measuring the amount of 4-pyren-1-ylbutanoic acid released by the enzyme with reversed-phase HPLC and fluorescence detection. The known FAAH inhibitors URB597, phenyl hexanoyl oxazolopyridine (PHOP) and [6-(2-methyl-4,5-diphenyl-1H-imidazol-1-yl)hexyl]carbamic acid phenyl ester were used to validate the test assay.
Keywords: Fatty acid amide hydrolase; Fluorescence assay; Inhibitor screening

A rapid, sensitive, and specific method was developed and validated using liquid chromatography-tandem mass spectrometry for the simultaneous quantitation of atorvastatin (ATV) and its major metabolite ortho-hydroxyatorvastatin (o-HATV) in human plasma. The sample preparation involved a liquid–liquid extraction without chlorinated solvents and an on-line solid-phase extraction exploring the possibilities that anion exchange offers. The analytical method presented intraday and day-to-day variation below 10%; intraday and day-to-day accuracy stood between 94% and 105%; the limit of quantification was 0.1 ng/mL for ATV and 0.5 ng/mL for o-HATV; and the recovery was above 75% for both molecules. This method was applied successfully to quantitate ATV and o-HATV concentrations in an unstudied renal transplant recipient cohort treated with an immunosuppressive regime of tacrolimus and mycophenolic acid and a cohort of hypercholesterolemic patients included in the study as a control group. It can be used to evaluate patient adherence, drug–drug interactions, and pharmacokinetic/pharmacodynamic relationships. The results in our study showed that ATV and o-HATV levels in the renal transplant group were significantly increased (p < 0.001), compared to the hypercholesterolemic group.
Keywords: Atorvastatin; Ortho-hydroxyatorvastatin; Mass spectrometry; Liquid chromatography; Solid-phase extraction; Kidney transplanted recipients

Four chiral derivatizing reagents (CDR 1–4), namely, FDNP-l-Ala, FDNP-l-Val, FDNP-l-Phe, and FDNP-l-Leu, were synthesized using microwave (MW) irradiation by substituting one of the fluorine atoms in difluoro dinitro benzene (DFDNB) with l-Ala, l-Val, l-Phe, and l-Leu (CDR 1–4). The other set of CDRs, namely, FDNP-l-Phe-NH2, FDNP-l-Val-NH2, and FDNP-l-Leu-NH2, was also prepared. These reagents were used for synthesis of diastereomers of 18 proteinogenic and 08 non-proteinogenic amino acids, which were resolved by reversed-phase high-performance liquid chromatography using C18 column and gradient eluting mixture of aq.TFA and acetonitrile with UV detection at 340 nm. The reagents were used for resolution of a complex mixture of 18 racemic proteinogenic amino acids in a single chromatographic run of 65 min and to determine concentration of the d-amino acid in a solution of dl-amino acid. The resolution (R S) and selectivity (α) obtained for the two sets of diastereomers were compared among themselves and among the two groups. The method was validated for accuracy, precision, limit of detection (LOD), and limit of quantification. LOD is 0.001% impurity of d-enantiomer.
Keywords: Chiral separation; α-Amino acids; Chiral variants of Sanger’s reagent; Liquid chromatography

Enhanced electrophoretic DNA separation in photonic crystal fiber by Yi Sun; Nam-Trung Nguyen; Yien Chian Kwok (1707-1710).
Joule heating generated by the electrical current in capillary electrophoresis leads to a temperature gradient along the separation channel and consequently affects the separation quality. We describe a method of reducing the Joule heating effect by incorporating photonic crystal fiber into a micro capillary electrophoresis chip. The photonic crystal fiber consists of a bundle of extremely narrow hollow channels, which ideally work as separation columns. Electrophoretic separation of DNA fragments was simultaneously but independently carried out in 54 narrow capillaries with a diameter of 3.7 μm each. The capillary bundle offers more efficient heat dissipation owing to the high surface-to-volume ratio. Under the same electrical field strength, notable improvement in resolution was obtained in the capillary bundle chip.
Keywords: Joule heating; Capillary bundle; Photonic crystal fiber; Microchip capillary electrophoresis; DNA separation

In this work, we have studied the effect of different probe lengths and surface densities on the hybridization of a 181-bp polymerase chain reaction product to probes tethered onto magnetic microparticles. Hybridization was shown to be favored by longer probes but only at probe surface densities where probe-to-probe interactions are absent. From these results, a simple rule was inferred for determining maximum surface densities above which hybridization signals decreased. According to this rule, if the average surface area occupied by an immobilized probe (Σ) is larger than the projected surface area of each tethered probe molecule (S ss ), hybridization efficiency increases with surface density, whereas the reverse occurs when Σ − S ss  < 0.
Keywords: DNA–DNA hybridization; Magnetic microparticles; Surface density; Probe–probe interactions