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Analytical and Bioanalytical Chemistry (v.400, #5)

Chromatographic party in Valencia (ISC 2010) by Yolanda Pico; Joan O. Grimalt (pp. 1197-1198).

has been a full professor of nutrition and food science at the University of Valencia since 1998. Her research interests include identification of unknown compounds by liquid chromatography–mass spectrometry, microextraction separations and environmental and food safety. She is the author of nearly 180 peer-reviewed papers, 140 scientific papers in journals and 25 book chapters and is the editor of three books on food and environmental safety and applications of liquid chromatography–mass spectrometry. Presently, she is Vice-president of the Spanish Society of Chromatography and Related Techniques (SECyTA). is a research professor at and the director of the Institute of Environmental Assessment and Water Research (IDÆA) of the Spanish Council for Scientific Research (CSIC). His professional activity is within environmental organic geochemistry. More specifically, his research is devoted to the study of natural and anthropogenic organic compounds as markers of the health status of ecosystems and living organisms (including humans). To date he has published about 520 scientific papers, most of them in international peer-reviewed journals. Presently, he is President of the Spanish Society of Chromatography and Related Techniques. He received the King Jaume I award devoted to the preservation of the 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).

New extraction sorbent based on aptamers for the determination of ochratoxin A in red wine by Florence Chapuis-Hugon; Aude du Boisbaudry; Benjamin Madru; Valérie Pichon (pp. 1199-1207).

A new solid phase extraction method based on aptamers, an oligosorbent, was developed and applied to the determination of ochratoxin A (OTA) from red wine. Two solid supports were chosen to immobilize OTA aptamer by covalent binding (cyanogen bromide-activated sepharose) or noncovalent binding (streptavidin-activated agarose). The resulting oligosorbents were evaluated in terms of retention, selectivity, and capacity. To assess the selectivity of the resulting oligosorbents, control supports made only of a solid support without immobilized aptamers were simultaneously studied. After optimization of the selective extraction procedure, extraction recoveries close to 100% were obtained on both materials. No retention was observed on the control supports. A similar capacity was also found for both oligosorbents. However, the immobilization by covalent bonding appeared more robust for the determination of OTA in the wine. A conventional sorbent and an immunoaffinity column were also applied to the determination of OTA in red wine to compare the potential of the various approaches for the treatment of such complex samples.

Keywords: Selective SPE; Aptamer; Oligosorbent; Wine; Ochratoxin A; Immobilization

Evaluation of new ionic liquids as high stability selective stationary phases in gas chromatography by Jaime González Álvarez; Domingo Blanco Gomis; Pilar Arias Abrodo; Daniel Díaz Llorente; Eduardo Busto; Nicolás Ríos Lombardía; Vicente Gotor Fernández; María Dolores Gutiérrez Álvarez (pp. 1209-1216).

Two ionic liquids (ILs), namely (S,S)-1-butyl-3-(2′-hydroxy-cyclohexyl)-3H-imidazol-1-ium tetrafluoroborate and (S,S)-1-butyl-3-(2′-acetyl-cyclohexyl)-3H-imidazol-1-ium tetrafluoroborate have been employed as stationary phases in capillary gas chromatography. These new phases exhibit a column efficiency of 1,600 and 2,100 plates m⁻¹ for IL 1 and IL 2, respectively, a wide operating temperature range and good thermal stability (bleeding temperature of 250 °C for IL 1 and 160 °C for IL 2). Inverse gas chromatography (GC) analyses were used to study the solvation properties of these ILs through a linear solvation energy model. The application of these ILs as new GC stationary phases was studied. These stationary phases exhibited unique selectivity for many organic substances, such as alkanes, ketones, esters, and aromatic compounds. The efficient separation of several mixtures containing compounds of different polarities and the good separation of fatty acid methyl esters (FAMEs) and cis/trans isomers indicate that these ILs may be applicable as a new type of GC stationary phases.

Keywords: Gas chromatography; Ionic liquids; Stationary phases

Optimal experimental designs in RPLC at variable solvent content and pH based on prediction error surfaces by J. R. Torres-Lapasió; S. Pous-Torres; J. J. Baeza-Baeza; M. C. García-Álvarez-Coque (pp. 1217-1230).

When pH is used as factor in reversed-phase liquid chromatographic (RPLC) separations, the need for providing quality and informative data with the minimal experimental effort becomes imperative. The most rational way to achieve this is by means of experimental designs. The interest in finding optimal designs involving solvent content and pH in RPLC is considerable, since these factors allow large variations in selectivity when ionisable compounds are involved. Unfortunately, the equations that describe the retention of these compounds with pH are nonlinear. As a consequence, factorial and other designs based on geometrical considerations are not well suited, whereas D-optimal and related designs can only be applied in an iterative fashion. In this work, an extension of G-optimal designs, aimed to enhance the quality of the predictions, is examined for problems involving solvent content and pH. The study was carried out with a set of probe ionisable compounds, for which information on retention behaviour was accurately known. A stepwise strategy was used to obtain a rapid estimation of the best design with a given number of experiments. The objective of the study was to investigate the distribution and number of points in the ideal design for compounds of different acid–base behaviour, and the possibility of finding common designs for groups of compounds. A further goal was to derive design construction rules containing the information requirements, without needing any further mathematical treatment. Figure Three-dimensional view of an error surface

Keywords: Chemometrics/statistics; HPLC; Modelling; Separation theory

Ligand exchange chromatography: a vital dimension for the reliable characterization of heterocycles in crude oils and refined products by Saroj K. Panda; Adnan A. Al-Hajji; Hendrik Müller; Omer R. Koseoglu (pp. 1231-1239).

In the present study, we established a statistical distribution pattern of indigenous sulfur, nitrogen, and oxygen species in Arabian Heavy crude oil and its distilled fractions: naphtha, gas oil, and vacuum gas oil (VGO) using chemical derivatization with methyl iodide and subsequent characterization by positive electrospray Fourier transform mass spectrometry. It was observed that sulfur species for naphtha and gas oil were accumulated at lower double bond equivalent values and at lower carbon numbers compared to VGO, whereas crude oil encompassed a complete range of the sulfur species detected in all distilled fractions. Moreover, the use of alumina column chromatography and ligand exchange chromatography (LEC) on a palladium-bonded silica stationary phase revealed additional structural features of sulfur heterocycles in terms of condensed and non-condensed thiophenes. During LEC separation, in addition to sulfur heterocycles, interesting results were obtained for oxygen-containing compounds. Ortho-substituted alkyl phenols were separated from meta- and para-substituted alkyl phenols on a palladium-bonded silica stationary phase.

Keywords: Crude oil; Phenols; Sulfur heterocycles; Normal phase chromatography; Hydrocarbons; Mass spectrometry

Capillary monolithic titania column for miniaturized liquid chromatography and extraction of organo-phosphorous compounds by Maguy Abi Jaoudé; Jérôme Randon (pp. 1241-1249).

A new sol–gel protocol was designed and optimized to produce titanium-dioxide-based columns within confined geometries such as monolithic capillary columns and porous-layer open-tubular columns. A surface pre-treatment of the capillary enabled an efficient anchorage of the monolith to the silica capillary wall during the synthesis. The monolith was further synthesized from a solution containing titanium n-propoxide, hydrochloric acid, N-methylformamide, water, and poly(ethylene oxide) as pore template. The chromatographic application of capillary titania-based columns was demonstrated with the separation of a set of phosphorylated nucleotides as probe molecules using aqueous normal-phase liquid chromatography conditions. Capillary titania monoliths offered a compromise between the high permeability and the important loading capacity needed to potentially achieve miniaturized sample preparations. The specificity of the miniaturized titania monolithic support is illustrated with the specific enrichment of 5′-adenosine mono-phosphate. The monolithic column offered a ten times higher loading capacity of 5′-adenosine mono-phosphate compared with that of the capillary titania porous-layer open-tubular geometry.

Keywords: Titania; Monolith; Sol–gel synthesis; Capillary column; Aqueous normal-phase chromatography; Phosphate enrichment; Phosphorylated compounds

Comparison of different types of stationary phases for the analysis of soy isoflavones by HPLC by N. Manchón; M. D’Arrigo; A. García-Lafuente; E. Guillamón; A. Villares; J. A. Martínez; A. Ramos; M. A. Rostagno (pp. 1251-1261).

Nowadays, there are new technologies in high-performance liquid chromatography columns available enabling faster and more efficient separations. In this work, we compared three different types of columns for the analysis of main soy isoflavones. The evaluated columns were a conventional reverse phase particle column, a fused-core particle column, and a monolithic column. The comparison was in terms of chromatographic parameters such as resolution, asymmetry, number of theoretical plates, variability of retention time, and peak width. The lower column pressure was provided by the monolithic column, although lower chromatographic performance was achieved. Conventional and fused-core particle columns presented similar pressure. Results also indicate that direct transfer between particle and monolithic columns is not possible requiring adjustment of conditions and a different method optimization strategy. The best chromatographic performance and separation speed were observed for the fused-core particle column. Also, the effect of sample solvent on the separation and peak shape was evaluated and indicated that monolithic column is the most affected especially when using higher concentrations of acetonitrile or ethanol. Sample solvent that showed the lowest effect on the chromatographic performance of the columns was methanol. Overall evaluation of methanol and acetonitrile as mobile phase for the separation of isoflavones indicated higher chromatographic performance of acetonitrile, although methanol may be an attractive alternative. Using acetonitrile as mobile phase resulted in faster, higher resolution, narrower, and more symmetric peaks than methanol with all columns. It also generated the lower column pressure and flatter pressure profile due to mobile phase changes, and therefore, it presents a higher potential to be explored for the development of faster separation methods.

Keywords: Soybean; Isoflavones; Analysis; Fused-core; Monolithic; Mobile phase; Sample solvent

Analytical characterization of mannosylerythritol lipid biosurfactants produced by biosynthesis based on feedstock sources from the agrofood industry by Matthias Onghena; Tinne Geens; Eliane Goossens; Marc Wijnants; Yolanda Pico; Hugo Neels; Adrian Covaci; Filip Lemiere (pp. 1263-1275).

Mannosylerythritol lipids (MELs) are currently one of the most promising biosurfactants because of their multifunctional applications and good biodegradability. Depending on the yeast strain and the feedstock used for the fermentation process, structural variations in the MELs obtained occur. Therefore, MELs produced by Pseudozyma aphidis DSMZ 70725 with a soybean oil feedstock were characterized by chromatography and mass spectrometry (MS). Column chromatography with silica provided fractionation of the different types of MEL. High-performance liquid chromatography combined with MS was employed for the analysis of the MEL fractions and crude mixtures. A characteristic MS pattern for the MELs was obtained and indications of the presence of new MEL homologues, showing the incorporation of longer and more unsaturated fatty acid chains than previously reported, were given. Gas chromatography–MS analysis confirmed the presence of such unsaturated fatty acid chains in the MELs, demonstrating the incorporation of fatty acids with lengths ranging from C₈ to C₁₄ and with up to two unsaturations per chain. The incorporation of C₁₆ and C₁₈ fatty acid chains requires further investigation. MS/MS data allowed the unambiguous identification of the fatty acids present in the MELs. The product ion spectra also revealed the presence of a new isomeric class of MELs, bearing an acetyl group on the erythritol moiety.

Keywords: Mannosylerythritol lipids; Mass spectrometry; Fatty acids; Liquid chromatography; Gas chromatography

Analysis of perfluorinated alkyl substances in Spanish sewage sludge by liquid chromatography–tandem mass spectrometry by Irene Navarro; Paloma Sanz; María Ángeles Martínez (pp. 1277-1286).

The present article describes the development of an analytical method for the determination of 13 perfluorinated alkyl substances (PFAS), as well as its application to real sewage sludge samples to confirm the presence of these compounds. The isolation of the analytes was performed by agitation, sonication and centrifugation techniques, followed by EnviCarb cleanup and weak anion exchange solid-phase extraction. Sensitive and selective determination was carried out by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS). Six mass-labelled internal standards were used to ensure the accuracy of the analytical results following isotopic dilution method. Several mobile phases (acetonitrile, methanol, mixtures of both and water with ammonium acetate or acetic acid) have been tested to reach the best resolution and reproducibility results. Other parameters related to MS/MS conditions were optimized. The reliability of the method was confirmed by the evaluation of linearity (R ² = 0.995–0.999), accuracy (84–99%) and injection repeatability and reproducibility (relative standard deviation below 19 and 23%, respectively). Limits of detection ranged from 0.007 to 2.217 pg. Recoveries show values higher than 80% for most of the target compounds. The application of this method to twenty real samples demonstrates its efficiency and accuracy, as well as provides for the first time to our knowledge, PFAS levels in sewage sludges from Spain. Figure Relative composition of individual PFAS in sewage sludge.

Keywords: Perfluorinated alkyl substances (PFAS); PFOS; PFOA; HPLC-MS/MS; Sewage sludge

Assessment of the occurrence and distribution of pharmaceuticals in a Mediterranean wetland (L’Albufera, Valencia, Spain) by LC-MS/MS by Pablo Vazquez-Roig; Vicente Andreu; Matthias Onghena; Cristina Blasco; Yolanda Picó (pp. 1287-1301).

The distribution of 17 pharmaceuticals between water and the solid phase (sediments and soils) was studied by utilizing solid-phase extraction (SPE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS). Two extraction procedures for soils and sediments, prior to the SPE, one based on pressurized liquid extraction (PLE) with hot water and the other on methanol/water ultrasonic extraction, were compared. Absolute recoveries were 71.2–99.3% [relative standard deviation (RSD) <21.4%)] for water, and the method detection limits (MDLs) ranged from 0.3 to 10 ng L⁻¹. Recoveries were 35.4–105.3% (RSDs <19.1%) and 42.1–97.8% (RSDs <14%) for soil and sediment samples, respectively, using PLE and 20.2–86.5% (RSDs <25.1%) and 30.3–97.4% (RSDs <19.1%) using ultrasonic extraction. Fifteen of the 17 pharmaceuticals were present in the L’Albufera water at concentrations up to 17 μg L⁻¹. Oxytetracycline and tetracycline were not detected. In sediments, only tetracycline, norfloxacin and diclofenac were not found. The other studied pharmaceuticals were present in the range from less than the method quantification limit (MQL) to 35.83 ng g⁻¹. Among the 17 target compounds, ofloxacin, ciprofloxacin, norfloxacin, trimethoprim, clofibric acid and diclofenac were not detected in soil samples. The average concentrations ranged from less than the MQL for ibuprofen to 34.91 ng g⁻¹ for tetracycline. These results indicate that pharmaceuticals could survive the wastewater treatment processes, which could lead to their dissemination in water environments.

Keywords: Pharmaceutical products; Surface water; Soil; Sediments; Wetlands; LC-MS/MS; Pressurized liquid extraction; Ultrasonic extraction; SPE

Quinolones control in milk and eggs samples by liquid chromatography using a surfactant-mediated mobile phase by M. Rambla-Alegre; M. A. Collado-Sánchez; J. Esteve-Romero; S. Carda-Broch (pp. 1303-1313).

Four quinolones (danofloxacin, difloxacin, flumequine and marbofloxacin) were determined in milk and egg samples by a simplified high-performance liquid chromatographic procedure using a micellar mobile phase. No extraction was needed to precipitate the proteins from the matrices since they were solubilised in micelles. The only pretreatment steps required were homogenisation, dilution and filtration before injecting the sample into the chromatographic system. An adequate resolution of the quinolones was achieved by a chemometrics approach where retention was modelled as a first step using the retention factors in only five mobile phases. Afterwards, an optimisation criterion was applied to consider the position and shape of the chromatographic peaks. Analytical separation involved a C18 reversed-phase column, a hybrid micellar mobile phase of 0.05 M sodium dodecyl sulphate, 10% (v/v) butanol and 0.5% (v/v) triethylamine buffered at pH 3 and fluorimetric detection. Quinolones were eluted in less than 15 min without the protein band or other endogenous compounds from the food matrices interfering. The calculated relevant validation parameters, e.g., decision limit (CC_α), detection capability (CC_β), repeatability, within-laboratory reproducibility, recoveries and robustness, were acceptable and complied with European Commission Decision 2002/657/EC. Finally, the proposed method was successfully employed in quantifying the four quinolones in spiked egg and milk samples.

Keywords: Quinolone; Micellar liquid chromatography; Food samples; Egg; Milk; Validation

An evaluation method for determination of non-polar pesticide residues in animal fat samples by using dispersive solid-phase extraction clean-up and GC-MS by Mercedes Castillo; Carmen González; Ana Miralles (pp. 1315-1328).

A rapid and easy method has been proposed, optimized and evaluated for quantitative determination at trace level of a representative group of non-polar pesticides in fat samples. The method includes n-hexane-saturated acetonitrile extraction, fat precipitation by cooling pre clean-up followed by dispersive solid-phase extraction (d-SPE) based on QuEChERS procedure clean-up. Determination was performed by gas chromatography–mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode. Efficiency of the d-SPE clean-up step was evaluated by comparison with fat oxidation treatment and gel permeation chromatography. Different combinations of d-SPE extraction reagents and sample amounts were tested in order to minimize matrix co-extractives and interferences. Best recoveries were obtained with 1200 mg of MgSO₄, 400 mg of end-capped C₁₈, 400 mg of PSA and 1 g of sample amount. SIM method, matrix effect, precision, and accuracy were evaluated with spiked pork fat samples for 38 representative pesticides. Results of this study showed that this technique is applicable in routine analysis for its application into monitoring programs. It simplifies time-consuming clean-up steps and allows a satisfactory long-term chromatographic performance.

Keywords: Pesticide; GC-MS; Dispersive solid-phase extraction; Animal fat; Optimization; QuEChERS

Use of dispersive liquid–liquid microextraction for the determination of carbamates in juice samples by sweeping-micellar electrokinetic chromatography by David Moreno-González; Laura Gámiz-Gracia; Ana M. García-Campaña; Juan M. Bosque-Sendra (pp. 1329-1338).

Dispersive liquid–liquid microextraction (DLLME) has been proposed for the extraction and preconcentration of 12 carbamate pesticides in juice samples, followed by their determination by micellar electrokinetic chromatography with diode-array detection. To improve sensitivity, an on-capillary sample concentration method based on sweeping has been developed. Also, separations were performed in an extended light path fused-silica capillary; the separation buffer consisted of 100 mM borate and 50 mM SDS (pH 9.0) with 5% acetonitrile. Samples were introduced by hydrodynamic injection, dissolved in the separation buffer, but free of micelles. Several parameters of the DLLME procedure (such as type and volume of extraction and dispersive solvents, pH, salt addition, and extraction time) were optimized. Recoveries obtained for fortified juice samples (banana, pineapple, and tomato) at three different concentration levels, ranged from 78% to 105%, with relative standard deviations lower than 9%. The limits of detection ranged from 1 to 7 μg l⁻¹. Moreover, the method is fast, simple, and environmentally friendly.

Keywords: Micellar electrokinetic chromatography (MEKC); Sweeping; Dispersive liquid–liquid microextraction (DLLME); Carbamates; Juices

Determination of carotenoids by liquid chromatography/mass spectrometry: effect of several dopants by S. Rivera; F. Vilaró; R. Canela (pp. 1339-1346).

Various carotenoids were analyzed by ultra-high-pressure liquid chromatography with tandem mass spectrometry detection (UHPLC-MS/MS). Three different techniques to ionize the carotenoids were compared: electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). For all of the carotenoids tested, it was possible to obtain characteristic transitions for their unequivocal identification using each ionization technique. APCI was shown to be a more powerful technique to ionize the carotenoids than ESI or APPI. Transitions to differentiate carotenoids that coelute were determined to distinguish antheraxanthin from astaxanthin and lutein from zeaxanthin. In addition, four dopants were evaluated to improve ionization and enhance the carotenoid signal strength in APPI. These dopants were acetone, toluene, anisole, and chlorobenzene. Carotenoids improved their response in almost all cases when a dopant was used. The use of dopants allowed the enhancement of the carotenoid signals strength up to 178-fold.

Keywords: Carotenoids; Atmospheric pressure photoionization (APPI); Dopant-assisted APPI (DA-APPI); Electrospray ionization (ESI); Atmospheric pressure chemical ionization (APCI)

Hydrodynamic and electrical considerations in the design of a four-electrode impedance-based microfluidic device by Gusphyl Justin; Mansoor Nasir; Frances S. Ligler (pp. 1347-1358).

A four-electrode impedance-based microfluidic device has been designed with tunable sensitivity for future applications to the detection of pathogens and functionalized microparticles specifically bound to molecular recognition molecules on the surface of a microfluidic channel. In order to achieve tunable sensitivity, hydrodynamic focusing was employed to confine the electric current by simultaneous introduction of two fluids (high- and low-conductivity solutions) into a microchannel at variable flow-rate ratios. By increasing the volumetric flow rate of the low-conductivity solution (sheath fluid) relative to the high-conductivity solution (sample fluid), increased focusing of the high-conductivity solution over four coplanar electrodes was achieved, thereby confining the current during impedance interrogation. The hydrodynamic and electrical properties of the device were analyzed for optimization and to resolve issues that would impact sensitivity and reproducibility in subsequent biosensor applications. These include variability in the relative flow rates of the sheath and sample fluids, changes in microchannel dimensions, and ionic concentration of the sample fluid. A comparative analysis of impedance measurements using four-electrode versus two-electrode configurations for impedance measurements also highlighted the advantages of using four electrodes for portable sensor applications. A four-electrode sensor with hydrodynamic focusing to confine that the current was characterized for tunable sensitivity

Keywords: Hydrodynamic focusing; Impedance spectroscopy; Nyquist plot; Bode impedance; Bode phase; Microfluidics

Hydrodynamic and electrical considerations in the design of a four-electrode impedance-based microfluidic device by Gusphyl Justin; Mansoor Nasir; Frances S. Ligler (pp. 1347-1358).

Keywords: Hydrodynamic focusing; Impedance spectroscopy; Nyquist plot; Bode impedance; Bode phase; Microfluidics

Development of an online SPE–LC–MS-based assay using endogenous substrate for investigation of soluble epoxide hydrolase (sEH) inhibitors by Nils Helge Schebb; Marion Huby; Christophe Morisseau; Sung Hee Hwang; Bruce D. Hammock (pp. 1359-1366).

Soluble epoxide hydrolase (sEH) is a promising therapeutic target for the treatment of hypertension, pain, and inflammation-related diseases. In order to enable the development of sEH inhibitors (sEHIs), assays are needed for determination of their potency. Therefore, we developed a new method utilizing an epoxide of arachidonic acid (14(15)-EpETrE) as substrate. Incubation samples were directly injected without purification into an online solid phase extraction (SPE) liquid chromatography electrospray ionization tandem mass spectrometry (LC–ESI–MS–MS) setup allowing a total run time of only 108 s for a full gradient separation. Analytes were extracted from the matrix within 30 s by turbulent flow chromatography. Subsequently, a full gradient separation was carried out on a 50X2.1 mm RP-18 column filled with 1.7 μm core–shell particles. The analytes were detected with high sensitivity by ESI–MS–MS in SRM mode. The substrate 14(15)-EpETrE eluted at a stable retention time of 96 ± 1 s and its sEH hydrolysis product 14,15-DiHETrE at 63 ± 1 s with narrow peak width (full width at half maximum height: 1.5 ± 0.1 s). The analytical performance of the method was excellent, with a limit of detection of 2 fmol on column, a linear range of over three orders of magnitude, and a negligible carry-over of 0.1% for 14,15-DiHETrE. The enzyme assay was carried out in a 96-well plate format, and near perfect sigmoidal dose–response curves were obtained for 12 concentrations of each inhibitor in only 22 min, enabling precise determination of IC₅₀ values. In contrast with other approaches, this method enables quantitative evaluation of potent sEHIs with picomolar potencies because only 33 pmol L⁻¹ sEH were used in the reaction vessel. This was demonstrated by ranking ten compounds by their activity; in the fluorescence method all yielded IC₅₀ ≤ 1 nmol L⁻¹. Comparison of 13 inhibitors with IC₅₀ values >1 nmol L⁻¹ showed a good correlation with the fluorescence method (linear correlation coefficient 0.9, slope 0.95, Spearman’s rho 0.9). For individual compounds, however, up to eightfold differences in potencies between this and the fluorescence method were obtained. Therefore, enzyme assays using natural substrate, as described here, are indispensable for reliable determination of structure–activity relationships for sEH inhibition.

Keywords: Soluble epoxide hydrolase (sEH); Natural substrate enzyme assay; Enzyme inhibitors turbulent-flow chromatography; Online-solid phase extraction; Liquid chromatography; Electrospray mass spectrometry tandem mass spectrometry

Toxicity of amorphous silica nanoparticles on eukaryotic cell model is determined by particle agglomeration and serum protein adsorption effects by Daniela Drescher; Guillermo Orts-Gil; Gregor Laube; Kishore Natte; Rüdiger W. Veh; Werner Österle; Janina Kneipp (pp. 1367-1373).

Cell cultures form the basis of most biological assays conducted to assess the cytotoxicity of nanomaterials. Since the molecular environment of nanoparticles exerts influence on their physicochemical properties, it can have an impact on nanotoxicity. Here, toxicity of silica nanoparticles upon delivery by fluid-phase uptake is studied in a 3T3 fibroblast cell line. Based on XTT viability assay, cytotoxicity is shown to be a function of (1) particle concentration and (2) of fetal calf serum (FCS) content in the cell culture medium. Application of dynamic light scattering shows that both parameters affect particle agglomeration. The DLS experiments verify the stability of the nanoparticles in culture medium without FCS over a wide range of particle concentrations. The related toxicity can be mainly accounted for by single silica nanoparticles and small agglomerates. In contrast, agglomeration of silica nanoparticles in all FCS-containing media is observed, resulting in a decrease of the associated toxicity. This result has implications for the evaluation of the cytotoxic potential of silica nanoparticles and possibly also other nanomaterials in standard cell culture.

Keywords: Agglomeration; Cytotoxicity; Fibroblast cells; Serum proteins; Silica nanoparticles

A new approach for generic screening and quantitation of potential genotoxic alkylation compounds by pre-column derivatization and LC-MS/MS analysis by A. M. van Wijk; B. Beerman; H. A. G. Niederländer; A. H. G. Siebum; G. J. de Jong (pp. 1375-1385).

A generic LC-MS/MS method was developed for the analysis of potentially genotoxic alkyl halides. A broad selection of alkyl halides were derivatized using 4-dimethylaminopyridine in acetonitrile. The reaction conditions for derivatization, i.e., solvent, reaction time, temperature and concentration of alkyl halide, active pharmaceutical ingredient (API), and reagent, were optimized for sensitivity and robustness. The interference of the matrix and the API and the presence of water on the derivatization reaction were investigated for a model drug product (paracetamol/caffeine tablets). Hydrophilic interaction liquid chromatography was used to allow a quantitative determination of the derivatives by tandem mass spectrometry. The derivatization reaction was shown to be selective for alkyl halides, although some reactivity was also observed for an aromatic sulfonate, which is also genotoxic. Even though differences in reaction efficiencies have been observed, the enhanced sensitivity obtained by the derivatization allows the majority of the alkyl halides to be detected by MS/MS at relevant levels for genotoxic impurity evaluation, i.e., 10 mg kg⁻¹. Another key advantage is that for the majority of derivatives, reagent-related fragments are produced, which allows low-level screening for alkyl halides. Highly specific MS detection can be performed using neutral loss and precursor ion scan experiments. The applicability of a generic screening method will make the genotox evaluation less dependent on the quality of assessments based on predictions only, and it will provide essential information during the development of new chemical entities. In addition to screening, target analysis in the low milligrams per kilogram range can be performed. A similar response of the derivatized compounds was obtained in the range of 1–100 mg kg⁻¹ with a reproducibility better than 10%, which is sufficient for the determination of alkyl halides in APIs and drug products.

Keywords: Genotoxic compounds; Alkyl halides; Derivatization; 4-Dimethylaminopyridine; Mass spectrometry; Neutral loss

Detection of DNA hybridisation in a diluted serum matrix by surface plasmon resonance and film bulk acoustic resonators by Sanna Auer; Martin Nirschl; Matthias Schreiter; Inger Vikholm-Lundin (pp. 1387-1396).

Nanomolar quantities of single-stranded DNA products ∼100 nucleotides long can be detected in diluted 1% serum by surface plasmon resonance (SPR) and film bulk acoustic resonators (FBARs). We have used a novel FBAR sensor in parallel with SPR and obtained promising results with both the acoustic and the optical device. Oligonucleotides and a repellent lipoamide, Lipa-DEA, were allowed to assemble on the sensor chip surfaces for only 15 min by dispensing. Lipa-DEA surrounds the analyte-binding probes on the surface and effectively reduces the non-specific binding of bovine serum albumin and non-complementary strands. In a highly diluted serum matrix, the non-specific binding is, however, a hindrance, and the background response must be reduced. Nanomolar concentrations of short complementary oligos could be detected in buffer, whereas the response was too low to be measured in serum. DNA strands that are approximately 100 base pairs long at concentrations as low as 1-nM could be detected both in buffer and in 1% serum by both SPR and the FBAR resonator. Figure A schematic drawing of the surface assembly of DNA probes and Lipa-DEA blocking molecules on the SPR-sensor/FBAR-resonator surface and the following binding of a single-stranded PCR product

Keywords: Surface plasmon resonance; Film bulk acoustic resonator; DNA hybridisation detection; Serum; Self-assembled monolayer; DNA sensor

Towards a synthetic avidin mimic by Jesper Wiklander; Björn C. G. Karlsson; Teodor Aastrup; Ian A. Nicholls (pp. 1397-1404).

A series of streptavidin-mimicking molecularly imprinted polymers has been developed and evaluated for their biotin binding characteristics. A combination of molecular dynamics and NMR spectroscopy was used to examine potential polymer systems, in particular with the functional monomers methacrylic acid and 2-acrylamidopyridine. The synthesis of copolymers of ethylene dimethacrylate and one or both of these functional monomers was performed. A combination of radioligand binding studies and surface area analyses demonstrated the presence of selectivity in polymers prepared using methacrylic acid as the functional monomer. This was predicted by the molecular dynamics studies showing the power of this methodology as a prognostic tool for predicting the behavior of molecularly imprinted polymers. The biotin binding characteristics of a series of molecularly imprinted polymers have been evaluated and correlated to predictions made by molecular dynamics simulations and ¹H-NMR titrations

Keywords: Molecularly imprinted polymer; Molecular dynamics; Receptor mimic; Biotin

An ultrasonication-assisted extraction and derivatization protocol for GC/TOFMS-based metabolite profiling by Yumin Liu; Tianlu Chen; Yunpin Qiu; Yu Cheng; Yu Cao; Aihua Zhao; Wei Jia (pp. 1405-1417).

Conventional chemical derivatization of metabolites in biological specimens is time-consuming, which limits the throughput and efficiency of metabolite profiling using a gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) platform. We report an ultrasonication-assisted protocol which reduces the derivatization time from hours to about 30 min and significantly enhances the derivatization efficiency prior to a GC/TOFMS analysis. The protocol was evaluated using 40 compounds representing different classes of human metabolites, and demonstrated good analytical precision and accuracy. In comparison with the conventional method, the new protocol was able to increase the intensity of most of the identified peaks (71.0%) in the GC/TOFMS chromatograms of human serum samples. The detected compounds with increased intensity include most amino acids, keto-containing organic acids, carbonyl-containing carbohydrates, and unsaturated fatty acids. We applied this protocol in a metabolomic study of human serum samples obtained from 34 patients diagnosed with hypertension and 29 age- and gender-matched healthy subjects. Metabolite markers associated with hypertension, including glucosamine, d-sorbitol, 1-stearoylglycerol, and homocysteine, were identified and validated by statistical methods and use of reference standards. Our work highlights the potential of this novel approach for the large-scale metabolite profiling of samples generated from plant, animal, and clinical and epidemiological studies.

Keywords: Metabolites; Metabolomics; Ultrasonication; Derivatization; Hypertension

Investigation of the applicability of Zernike moments to the classification of SDS 2D-PAGE maps by Emilio Marengo; Marina Cocchi; Marco Demartini; Elisa Robotti; Marco Bobba; Pier Giorgio Righetti (pp. 1419-1431).

The aim of this work is to investigate the performance of multivariate classification techniques like partial least squares–discriminant analysis (PLS-DA) and linear discriminant analysis (LDA) when using Zernike moments as global image descriptors, in the classification of sodium dodecyl sulphate (SDS) two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) maps affected by different levels of deformation. Synthetic sets of images simulating real SDS 2D-PAGE maps were analysed in controlled conditions to obtain information on the robustness and limits of applicability of the classification techniques operating on the basis of a given image decomposition method.

Keywords: Classification; SDS 2D-PAGE; Gel electrophoresis; Zernike moments

Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms by Andréia Z. Dinon; Theo W. Prins; Jeroen P. van Dijk; Ana Carolina M. Arisi; Ingrid M. J. Scholtens; Esther J. Kok (pp. 1433-1442).

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples.

Keywords: GMO; UGM; Real-time PCR; Cry genes

Dynamics of pistachio oils by proton nuclear magnetic resonance relaxation dispersion by Pellegrino Conte; Valerio Mineo; Salvatore Bubici; Claudio De Pasquale; Farid Aboud; Antonella Maccotta; Diego Planeta; Giuseppe Alonzo (pp. 1443-1450).

A number of pistachio oils were selected in order to test the efficacy of nuclear magnetic resonance relaxation dispersion (NMRD) technique in the evaluation of differences among oils (1) obtained from seeds subjected to different thermal desiccation processes, (2) retrieved from seeds belonging to the same cultivar grown in different geographical areas and (3) produced by using seed cultivars sampled in the same geographical region. NMRD measures relaxation rate values which are related to the dynamics of the chemical components of complex food systems. Results not only allowed to relate kinematic viscosity to relaxometry parameters but also were successful in the differentiation among the aforementioned oils. In fact, from the one hand, the larger the kinematic viscosity, the faster the rotational motions appeared as compared to the translational ones. On the other hand, relaxation rate curves (NMRD) varied according to the oxidative stresses and chemical composition of each sample. The present study showed for the first time that NMRD is a very promising technique for quick evaluations of pistachio oil quality without the need for time-consuming chemical manipulations.

Keywords: Pistachio oils; FFC-NMR; NMRD; Relaxometry; Kinematic viscosity

Reexamination of the ORAC assay: effect of metal ions by E. Nkhili; P. Brat (pp. 1451-1458).

The oxygen radical absorbance capacity (ORAC) assay method has been employed extensively in the field of antioxidant and oxidative stress. It uses fluorescein as probe for oxidation by peroxyl radical. Hundreds of reports have been published on the use of this method to determine antioxidant capacity in food and biological samples. The question is whether the results of all these reports are influenced by antioxidant autoxidation, which occurs during the ORAC test. Indeed, the presence of metal ions in the studied matrix will influence antioxidant stability, thereby leading to the underestimation of their antioxidant properties. Ethylenediaminetetraacetic acid hydrate (EDTA) can be used as a metal complexation agent. This paper examines the effect of the addition of EDTA on the ORAC values of pure compounds (quercetin, ascorbic, and dehydroascorbic acid) and five food juices (kiwi, orange, tomato, red grape, and apple). Metal complexation by EDTA (80 μM) clearly increased the ORAC values, given that the antioxidant was protected against rapid autoxidation incited by trace metal ions within samples and then by free radicals. Our finding also undoubtedly demonstrated that the number of literature values is potentially underestimated.

Keywords: ORAC; Antioxidant activity; Phenolic compounds; Metal chelator; Fruit juices

Development of analytical strategies using U-HPLC-MS/MS and LC-ToF-MS for the quantification of micropollutants in marine organisms by Klaas Wille; Julie A. L. Kiebooms; Michiel Claessens; Karen Rappé; Julie Vanden Bussche; Herlinde Noppe; Nander Van Praet; Eric De Wulf; Peter Van Caeter; Colin R. Janssen; Hubert F. De Brabander; Lynn Vanhaecke (pp. 1459-1472).

Organic micropollutants such as pharmaceuticals, perfluorinated compounds (PFCs), and pesticides, are important environmental contaminants. To obtain more information regarding their presence in marine organisms, an increasing demand exists for reliable analytical methods for quantification of these micropollutants in biotic matrices. Therefore, we developed extraction procedures and new analytical methods for the quantification of 14 pesticides, 10 PFCs, and 11 pharmaceuticals in tissue of marine organisms, namely blue mussels (Mytilus edulis). This paper presents these optimized analytical procedures and their application to M. edulis, deployed at five stations in the Belgian coastal zone. The methods consisted of a pressurized liquid extraction and solid-phase extraction (SPE) followed by ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry for pharmaceuticals and pesticides, and of a liquid extraction using acetonitrile and SPE, followed by liquid chromatography coupled to time-of-flight mass spectrometry for PFCs. The limits of quantification of the three newly optimized analytical procedures in M. edulis tissue varied between 0.1 and 10 ng g⁻¹, and satisfactory linearities (≥0.98) and recoveries (90–106%) were obtained. Application of these methods to M. edulis revealed the presence of five pharmaceuticals, two PFCs, and seven pesticides at levels up to 490, 5, and 60 ng g⁻¹, respectively. The most prevalent micropollutants were salicylic acid, paracetamol, perfluorooctane sulfonate, chloridazon, and dichlorvos.

Keywords: Pharmaceuticals; Perfluorinated compounds; Pesticides; Marine organisms; Liquid chromatography; Mass spectrometry

The identification of synthetic organic pigments in modern paints and modern paintings using pyrolysis-gas chromatography–mass spectrometry by Joanna Russell; Brian W. Singer; Justin J. Perry; Anne Bacon (pp. 1473-1491).

A collection of more than 70 synthetic organic pigments were analysed using pyrolysis-gas chromatography–mass spectrometry (Py-GC–MS). We report on the analysis of diketo-pyrrolo-pyrrole, isoindolinone and perylene pigments which are classes not previously reported as being analysed by this technique. We also report on a number of azo pigments (2-naphthol, naphthol AS, arylide, diarylide, benzimidazolone and disazo condensation pigments) and phthalocyanine pigments, the Py-GC–MS analysis of which has not been previously reported. The members of each class were found to fragment in a consistent way and the pyrolysis products are reported. The technique was successfully applied to the analysis of paints used by the artist Francis Bacon (1909–1992), to simultaneously identify synthetic organic pigments and synthetic binding media in two samples of paint taken from Bacon’s studio and micro-samples taken from three of his paintings and one painting attributed to him. Figure Dulux orange paint in the studio of Francis Bacon, now located at Dublin City Gallery, The Hugh Lane

Keywords: Py-GC–MS; Francis Bacon; Synthetic organic pigment; Pyrolysis; Art; Mass spectrometry

Ancient and historic steel in Japan, India and Europe, a non-invasive comparative study using thermal neutron diffraction by F. Grazzi; F. Civita; A. Williams; A. Scherillo; E. Barzagli; L. Bartoli; D. Edge; M. Zoppi (pp. 1493-1500).

The production and refinement of steel has followed very different paths in different parts of the Eurasian continent. In aiming to characterize the similarities and differences between various smelting and smithing methods, we have analysed steel samples from four different areas and historic periods: the Kotō Age in Japan (twelfth–sixteenth century), the Moghul Empire in India (seventeenth–nineteenth century), the Ottoman Turkish Empire (seventeenth century) and the late Middle Ages (fifteenth century) in Italy. The best quality steel was employed for forging arms and armour of high quality, so that we have selected samples from Japan, India, the Middle East and Italy belonging to such a category. Traditional methods, such as metallography, used to characterize different steels in terms of their carbon contents, microconstituents and slag inclusions, entailed an invasive approach. Since many of the selected artefacts are in a very good state of conservation, a different and non-invasive approach was desirable. To this aim, we have used time of flight neutron diffraction on the Italian Neutron Experimental Station diffractometer, located at the pulsed neutron source ISIS in the United Kingdom. By this technique, we were able to quantify the phase distribution of the metal phases, the slag inclusion content, and the oxidation state of the samples, both as average concentration on the whole artefact and in selected gauge volumes. The results of the present investigation offer an interesting picture of the steel metallurgy in different areas of the world.

Keywords: Neutron diffraction; Phase analysis; Quantitative analysis; Steel metallurgy; Carbon content; Japanese swords; Indian swords; Ottoman gun; Medieval Italian weapons

Extracting natural dyes from wool—an evaluation of extraction methods by Ana Manhita; Teresa Ferreira; António Candeias; Cristina Barrocas Dias (pp. 1501-1514).

The efficiency of eight different procedures used for the extraction of natural dyes was evaluated using contemporary wool samples dyed with cochineal, madder, woad, weld, brazilwood and logwood. Comparison was made based on the LC-DAD peak areas of the natural dye’s main components which had been extracted from the wool samples. Among the tested methods, an extraction procedure with Na₂EDTA in water/DMF (1:1, v/v) proved to be the most suitable for the extraction of the studied dyes, which presented a wide range of chemical structures. The identification of the natural dyes used in the making of an eighteenth century Arraiolos carpet was possible using the Na₂EDTA/DMF extraction of the wool embroidery samples and an LC-DAD-MS methodology. The effectiveness of the Na₂EDTA/DMF extraction method was particularly observed in the extraction of weld dye components. Nine flavone derivatives previously identified in weld extracts could be identified in a single historical sample, confirming the use of this natural dye in the making of Arraiolos carpets. Indigo and brazilwood were also identified in the samples, and despite the fact that these natural dyes were referred in the historical recipes of Arraiolos dyeing, it is the first time that the use of brazilwood is confirmed. Mordant analysis by ICP-MS identified the widespread use of alum in the dyeing process, but in some samples with darker hues, high amounts of iron were found instead.

Keywords: Natural dyes; Dye extraction; Historical textiles; Arraiolos carpets; LC-DAD-MS; ICP-MS

Investigation of novel sol–gel hydrophobic surfaces for desorption electrospray ionization-mass spectrometry analysis by Andrea Penna; Lisa Elviri; Maria Careri; Alessandro Mangia; Giovanni Predieri (pp. 1515-1523).

Sol–gel-based materials were synthesized, characterized and finally tested as solid supports for desorption electrospray ionization-mass spectrometry (DESI-MS) analysis of a mixture of compounds of different polarity. Films with thickness in the 2–4 μm range were obtained by a dip-coating process using tetraethoxysilane (TEOS) and octyltriethoxysilane (OTES) as sol–gel precursors. Three types of surface with different hydrophobic character were obtained by varying the TEOS/OTES ratio in the sol–gel mixture. Each coating was characterized by atomic force microscopy investigations, gaining insight into homogeneity, smoothness and thickness of the obtained films. To study hydrophobicity of each surface, surface free energy measurements were performed. Different DESI-MS responses were observed when different solvent mixture deposition procedures and solvent spray compositions were investigated. Results were finally compared to those obtained by using commercial polytetrafluoroethylene-coated slides. It was found that surface free energy plays an important role in the desorption/ionization process as a function of the polarity of analytes.

Keywords: Desorption electrospray ionization; Mass spectrometry; Sol–gel; Hydrophobic surface

Analysis of staphylococcal enterotoxin A in milk by matrix-assisted laser desorption/ionization-time of flight mass spectrometry by Isabel Sospedra; Carla Soler; Jordi Mañes; José Miguel Soriano (pp. 1525-1531).

Staphylococcal enterotoxin A (SEA) is an exotoxin excreted mainly by Staphylococcus aureus and nowadays is the most prevalent compound in staphylococcal food poisoning worldwide. SEA is highly heat-resistant, and usual cooking times and temperatures are unlikely to completely inactivate it. A procedure for extraction of this toxin based on protein precipitation with a mixture of dichloromethane and acidified water was used before SDS-PAGE separation of soluble proteins. Finally, bands of interest were excised from the gel and in-gel enzymatic digestion was done. SEA from pasteurized milk was detected with matrix-assisted laser-desorption/ionization–time of flight (MALDI–TOF) mass spectrometry. Nineteen peptides (range 800–2400 Da) were identified as products of trypsin cleavage of the SEA standard with a score of 204 and 73% coverage of the protein sequence, whereas thirteen peptides were revealed for SEA extracted from milk with a score of 148 and 58% sequence coverage obtained. This procedure has been applied successfully for identification of SEA in milk.

Keywords: MALDI–TOF; Milk; Staphylococcal enterotoxin A; Staphylococcus aureus ; Tryptic digestion

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Analytical and Bioanalytical Chemistry (v.400, #5)