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Analytical and Bioanalytical Chemistry (v.402, #7)

Bavarois challenge by Hervé This (pp. 2229-2230).

Solution to “little western flower” challenge by Reinhard Meusinger (pp. 2231-2232).

Application of medical and analytical methods in Lyme borreliosis monitoring by Magdalena Ligor; Paweł Olszowy; Bogusław Buszewski (pp. 2233-2248).

Lyme borreliosis (LB) is one of the most common tick-borne diseases in the northern hemisphere. It is a chronic inflammatory disease caused by the spirochaete Borrelia burgdorferi. In its early stages, pathological skin lesions, namely erythema chronicum migrans, appear. The lesions, usually localised at the site of the bite, may become visible from a few weeks up to 3 months after the infection. Predominant clinical symptoms of the disease also involve joint malfunctions and neurological or cardiac disorders. Lyme disease, in all its stages, may be successfully treated with antibiotics. The best results, however, are obtained in its early stages. In order to diagnose the disease, numerous medical or laboratory techniques have been developed. They are applied to confirm the presence of intact spirochaetes or spirochaete components such as DNA or proteins in tick vectors, reservoir hosts or patients. The methods used for the determination of LB biomarkers have also been reviewed. These biomarkers are formed during the lipid peroxidation process. The formation of peroxidation products generated by human organisms is directly associated with oxidative stress. Apart from aldehydes (malondialdehyde and 4-hydroxy-2-nonenal), many other unsaturated components such as isoprostenes and neuroprostane are obtained. The fast determination of these compounds in encephalic fluid, urine or plasma, especially in early stages of the disease, enables its treatment. Various analytical techniques which allow the determination of the aforementioned biomarkers have been reported. These include spectrophotometry as well as liquid and gas chromatography. The analytical procedure also requires the application of a derivatization step by the use of selected reagents.

Keywords: Lyme borreliosis; Biomarkers; Analytical methods; Malondialdehyde; 4-Hydroxy-2-nonenal; Prostanes

Application of microextraction by packed sorbent to isolation of psychotropic drugs from human serum by Renata Wietecha-Posłuszny; Aneta Garbacik; Michał Woźniakiewicz; Agnieszka Moos; Marcin Wieczorek; Paweł Kościelniak (pp. 2249-2257).

A method of microextraction by packed sorbent (MEPS) followed by liquid chromatography with diode array detection has been developed and optimized for the extraction of six tricyclic antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin) from human serum. The optimal parameters of MEPS extraction (type of sorbent, volume of sample, composition, and volume of washing and elution solutions) for these drugs in spiked samples were defined. The developed MEPS procedure was validated and then successfully applied to the analysis of serum reference material. The limit of detection (0.02–0.05 μg/mL), intraday (2.7–8.8%) and interday (4.4–11.6%) precision (RSD), and the accuracy of the assay (94.5–108.8%) at three concentration levels—0.2, 0.5, and 0.8 μg/mL—were estimated. The accuracy of the method was evaluated by the analysis of certified reference material. Moreover, the validated procedure was compared with the solid-phase extraction technique. Finally, microextraction by packed sorbent was assessed as a suitable tool in forensic and clinical methods for serum sample preparations.

Keywords: Tricyclic antidepressant drugs; Microextraction by packed sorbent; Solid-phase extraction; High-performance liquid chromatography; Human serum samples

Electrochemical uranyl biosensor with DNA oligonucleotides as receptor layer by Robert Ziółkowski; Łukasz Górski; Sławomir Oszwałdowski; Elżbieta Malinowska (pp. 2259-2266).

The feasibility of using gold electrodes modified with short-chain ssDNA oligonucleotides for determination of uranyl cation is examined. Interaction between UO ₂ ²⁺ and proposed recognition layer was studied by means of voltammetric and quartz crystal microbalance measurements. It was postulated that ssDNA recognition layer functions via strong binding of UO ₂ ²⁺ to phosphate DNA backbone. The methylene blue was used as a redox marker for analytical signal generation. Biosensor response was based on the difference in electrochemical signal before and after subjecting it to sample containing uranyl ion. The lower detection limit of 30 nmol L⁻¹ for UO ₂ ²⁺ was observed for a sample incubation time of 60 min. Proposed ssDNA-modified electrodes demonstrated good selectivity towards UO ₂ ²⁺ against common metal cations, with only Pb²⁺ and Ca²⁺ showing considerable interfering effect.

Keywords: Biosensors; Electroanalytical methods; Electrochemical sensors; Mass sensitive sensors; Stripping analysis

Synchrotron radiation Fourier-transform infrared and Raman microspectroscopy study showing an increased frequency of creatine inclusions in the rat hippocampal formation following pilocarpine-induced seizures by J. Dulinska; Z. Setkowicz; K. Janeczko; C. Sandt; P. Dumas; L. Uram; K. Gzielo-Jurek; J. Chwiej (pp. 2267-2274).

In the present work, synchrotron radiation Fourier-transform infrared (SRFTIR) and Raman microspectroscopies were used to evaluate a possible role of creatine in the pathogenesis and progress of pilocarpine-evoked seizures and seizure-induced neurodegenerative changes in the rat hippocampal tissue. The main goal of this study was to identify creatine deposits within the examined brain area, to analyze their frequency in epileptic animals and naive controls and to examine correlations between the number of inclusions in the hippocampal formation of epileptic rats and the quantitative parameters describing animal behavior during 6-h observation period after pilocarpine injection. The presence of creatine in the brain tissue was confirmed based on the vibrational bands specific for this compound in the infrared and Raman spectra. These were the bands occurring at the wavenumbers around 2800, 1621, 1398, and 1304 cm⁻¹ in IR spectra and around 1056, 908 and 834 cm⁻¹ in the Raman spectra. Creatine was detected in eight of ten analyzed epileptic samples and in only one of six controls under the study. The number of deposits in epileptic animals varied from 1 to 100 and a relative majority of inclusions were detected in the area of the Dentate Gyrus and in the multiform hippocampal layer. Moreover, the number of creatine inclusions was positively correlated with the total time of seizure activity.

Keywords: Pilocarpine-induced epilepsy; Creatine deposits; SRFTIR microspectroscopy and imaging; Raman microspectroscopy

Mass spectrometry: Fifth meeting of the Spanish Society of Mass Spectrometry (SEEM) by José M. Vadillo; Damià Barceló (pp. 2275-2276).

is Associate Professor at the Analytical Chemistry Department of the University of Málaga (Spain). He graduated in Biology at the University of Navarra and defended his PhD in Analytical Chemistry in the topic of laser-induced breakdown spectroscopy at the University of Malaga under the supervision of J.J. Laserna. After a Fulbright Fellow post-doc period at Richard Zare’s laboratory at Stanford University, he was recruited with a Ramón and Cajal research contract to the University of Malaga. His research interests mainly focus on the use of optical and mass spectrometry microprobes for direct condensed-phase analysis. is Research Professor of the Scientific Research Council (CSIC) of the Government of Spain, at the Institute of Environmental Assessment and Water Research (IDAEA), Barcelona, Spain, and Director of the Catalan Institute for Water Research (ICRA). His fields of specialization include water quality assessment and management; fate and behaviour of emerging contaminants in surface waters, wastewaters, and groundwater; and analysis, fate, and risk of nanomaterials in the environment. He has directed over thirty PhD students, and is regularly invited to hold short and continuous education courses at different universities, international institutions, and PITTCON. Since December 2009 he has been Visiting Professor at King Saud University, Riyadh, Saudi Arabia. In 2007 was awarded the King Jaime I Prize for the Protection of Nature by the Generalitat of Valencia, Spain.

Multi-analytical study of patination methods on steel substrates: a full insight into surface chemistry and morphology by H. Téllez; J. M. Vadillo; J. J. Laserna; R. J. Chater; D. S. McPhail (pp. 2277-2285).

Patination of metals has been used for decorative or protective purposes, and several methods aimed to create coloured films on metal surfaces have been developed. This work describes a multi-analytical approach to characterize artificial blue patinas created on mild steel substrates by means of traditional recipes and methods for colouring ancient objects and artefacts. We suggest the combined use of secondary ion mass spectrometry, focused ion beam, X-ray diffraction spectroscopy, white light interferometry and reflectance spectroscopy to characterize blue patinas on steel substrates and to investigate the relationship between the developed colour and the patina layer microstructure and composition. Therefore, the analysis of the oxide films produced by either thermal or chemical colouring methods has been successfully performed, providing information about the film morphology, the surface composition and in-depth elemental distribution within the coloured layers, and the origin of the colour developed on the surface. Figure Blue patina developed on a mild steel substrate using thermal colouring methods

Keywords: Colouring methods; Surface analysis; Iron oxides; Patinas; SIMS; FIB

Multi-residue determination of pesticides in tropical fruits using liquid chromatography/tandem mass spectrometry by A. M. Botero-Coy; J. M. Marín; M. Ibáñez; J. V. Sancho; F. Hernández (pp. 2287-2300).

Monitoring pesticide residues in tropical fruits is of great interest for many countries, e.g., from South America, that base an important part of their economy on the exportation of these products. In this work, a LC-MS/MS multi-residue method using a triple quadrupole analyzer has been developed for around 30 pesticides in seven Colombian tropical fruits of high commercial value for domestic and international markets (uchuva, tamarillo, granadilla, gulupa, maracuya, papaya, and pithaya). After sample extraction with acetonitrile, an aliquot of the extract was diluted with water and directly injected into the HPLC-MS/MS system (electrospray interface) without any cleanup step. The formation of sodium adducts—of poor fragmentation—was minimized using 0.1% formic acid in the mobile phase, which favored the formation of the protonated molecule. However, the addition of ammonium acetate made the formation of the ammonium adducts in some particular cases possible, avoiding the presence of the sodium adducts. The highest sensitivity was observed in positive electrospray ionization for the wide majority of pesticides, with a few exceptions for acidic compounds that gave better response in the negative mode (e.g., 2,4-D, fluazinan). Thus, simultaneous acquisition on the positive/negative mode was applied. Two MS/MS transitions were acquired for each compound to ensure a reliable quantification and identification of the compounds detected in samples, although for malathion a third transition was acquired due to the presence of interfering isobaric compounds in the sample extracts. A detailed study of matrix effects was made by a comparison of standards in solvent and in matrix. Both ionization suppression and ionization enhancement were observed depending on the analyte/matrix combination tested. Correction of matrix effects was made by the application of calibration in matrix. Three matrices were selected (uchuva, maracuya, gulupa) to perform matrix calibration in the analysis of all seven fruit varieties studied. The method was validated by recovery experiments in samples spiked at two levels (0.05 and 0.5 mg/kg). The data were satisfactory for the wide majority of analyte/matrix combinations, with most recoveries between 70% and 110% and the RSD below 15%. Several samples collected from the market were finally analyzed. Positive findings were confirmed by evaluating the experimental Q/q ratios and retention times, and comparing them with those of reference standards. Figure Tropical fruits investigated in this work and LC-MS/MS chromatograms illustrating the detection and confirmation of pyrimethanil in gulupa

Keywords: Tropical fruits; LC-MS/MS; Pesticide residue analysis; Multi-residue method; Matrix effects; Triple quadrupole

Multiclass determination of 66 organic micropollutants in environmental water samples by fast gas chromatography–mass spectrometry by Laura Cherta; Joaquim Beltran; Tania Portolés; Félix Hernández (pp. 2301-2314).

A multiresidue method has been developed for quantification and identification of 66 multiclass priority organic pollutants in water by fast gas chromatography (GC) coupled to mass spectrometry (MS). Capabilities and limitations of single quadrupole mass spectrometer as detector in fast GC were studied evaluating the chromatographic responses in terms of sensitivity and chromatographic peak shapes, as they were influenced by scan time. The number of monitored ions in a selected ion monitoring (SIM) group strongly conditioned the scan time and subsequently the number of data points per peak. A compromise between peak shape and scan time was adopted in order to reach the proper conditions for quantitative analysis. An average of 10–15 points per peak was attained for most compounds, involving scan times between 0.1 and 0.22 s. The method was validated for mineral, surface, and groundwater. A solid-phase extraction pre-concentration step using C₁₈ cartridges was applied. Four isotopically labeled standards were added to the samples before extraction and used as surrogates to ensure a reliable quantification. Analyses were performed by GC–MS in electron ionization mode, monitoring the three most abundant and/or specific ions for each compound and using the intensity ratios as a confirmatory parameter. With a chromatographic run of less than 10 min, SIM mode provided excellent sensitivity and identification capability due to the monitoring of three ions and the evaluation of their intensity ratio. Limits of detection below 10 ng/L were reached for most of the 66 compounds in the three matrices studied. Accuracy and precision of the method were evaluated by means of recovery experiments at two fortification levels (10 and 100 ng/L), obtaining recoveries between 70% and 120% in most cases and relative standard deviations below 20%. The possibilities of a simultaneous SIM scan method have also been explored for non-target qualitative analysis. The developed method has been applied to the analysis of surface water samples collected from the Mediterranean region of Spain. Figure Full scan chromatogram of a 100 ng/mL standard mixture in hexane obtained by GC-MS in full scan mode (upper) and graphical diagram of the analytical procedure

Keywords: Pesticides; Organic pollutants; Fast gas chromatography; Mass spectrometry; Water analysis

Headspace gas chromatography–mass spectrometry for rapid determination of halonitromethanes in tap and swimming pool water by I. Montesinos; M. Gallego (pp. 2315-2323).

Halonitromethanes (HNMs) are one of the most cytotoxic and genotoxic classes found among the unregulated disinfection by-products formed by the reaction of chemical disinfectants with natural organic matter in water. Typical methods used to determine these compounds in water (mainly trichloronitromethane) are based on the Environmental Protection Agency (EPA) method 551.1 using liquid–liquid extraction. A fast and straightforward method for the determination of the nine HNMs in water has been developed using a static headspace (HS) coupled with gas chromatography–mass spectrometry (GC-MS). Important parameters controlling headspace extraction were optimised to obtain the highest sensitivity: 250 μL of methyl tert-butyl ether (as a chemical modifier) and 6 g of anhydrous sodium sulphate were added to the water sample; an oven temperature of 80 °C and an equilibration time of 20 min were also selected. The addition of a chemical modifier favoured the volatilisation of all HNMs, increasing their signals up to approximately four times. Under optimum conditions, the method developed provides limits of detection between 0.03 and 0.60 μg/L and a relative standard deviation of ∼6.0%. The developed method was validated and then compared with the reference method EPA 551.1 for the analysis of tap and swimming pool water. A good agreement in the results was observed, which corroborated the good performance of the proposed HS-GC-MS method. Figure

Keywords: Static headspace technique; Gas chromatography–mass spectrometry; Halonitromethanes; Tap and swimming pool water analysis

Analysis of UV filters in tap water and other clean waters in Spain by M. Silvia Díaz-Cruz; Pablo Gago-Ferrero; Marta Llorca; Damià Barceló (pp. 2325-2333).

The present paper describes the development of a method for the simultaneous determination of five hormonally active UV filters namely benzophenone-3 (BP3), 3-(4-methylbenzylidene) camphor (4MBC), 2-ethylhexyl 4-(dimethylamino) benzoate (OD-PABA), 2-ethylhexyl 4-methoxycinnamate (EHMC) and octocrylene (OC) by means of solid-phase extraction and gas chromatography–electron impact ionization–mass spectrometry. Under optimized conditions, this methodology achieved low method limits of detection (needed for clean waters, especially drinking water analysis), between 0.02 and 8.42 ng/L, and quantitative recovery rates higher than 73% in all cases. Inter- and intraday precision for all compounds were lower than 7% and 11%, respectively. The optimized methodology was applied to perform the first survey of UV absorbing compounds in tap water from the metropolitan area and the city of Barcelona (Catalonia, Spain). In addition, other types of clean water matrices (mineral bottled water, well water and tap water treated with an ion-exchange resin) were investigated as well. Results evidenced that all the UV filters investigated were detected in the water samples analyzed. The compounds most frequently found were EHMC and OC. Maximum concentrations reached in tap water were 290 (BP3), 35 (4MBC), 110 (OD-PABA), 260 (EHMC), and 170 ng/L (OC). This study constitutes the first evidence of the presence of UV filter residues in tap water in Europe.

Keywords: Gas chromatography–mass spectrometry; Hormonally active compounds; Organic UV filters; Sunscreens; Tap water

Determination of glyphosate in groundwater samples using an ultrasensitive immunoassay and confirmation by on-line solid-phase extraction followed by liquid chromatography coupled to tandem mass spectrometry by Josep Sanchís; Lina Kantiani; Marta Llorca; Fernando Rubio; Antoni Ginebreda; Josep Fraile; Teresa Garrido; Marinella Farré (pp. 2335-2345).

Despite having been the focus of much attention from the scientific community during recent years, glyphosate is still a challenging compound from an analytical point of view because of its physicochemical properties: relatively low molecular weight, high polarity, high water solubility, low organic solvent solubility, amphoteric behaviour and ease to form metal complexes. Large efforts have been directed towards developing suitable, sensitive and robust methods for the routine analysis of this widely used herbicide. In the present work, a magnetic particle immunoassay (IA) has been evaluated for fast, reliable and accurate part-per-trillion monitoring of glyphosate in water matrixes, in combination with a new analytical method based on solid-phase extraction (SPE), followed by liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS), for the confirmatory analysis of positive samples. The magnetic particle IA has been applied to the analysis of about 140 samples of groundwater from Catalonia (NE Spain) collected during four sampling campaigns. Glyphosate was present above limit of quantification levels in 41% of the samples with concentrations as high as 2.5 μg/L and a mean concentration of 200 ng/L. Good agreement was obtained when comparing the results from IA and on-line SPE-LC-MS/MS analyses. In addition, no false negatives were obtained by the use of the rapid IA. This is one of the few works related to the analysis of glyphosate in real groundwater samples and the presented data confirm that, although it has low mobility in soils, glyphosate is capable of reaching groundwater.

Keywords: Glyphosate; Groundwater; ELISA; Immunoassay; On-line SPE; LC-MS/MS

Determination of parabens and endocrine-disrupting alkylphenols in soil by gas chromatography–mass spectrometry following matrix solid-phase dispersion or in-column microwave-assisted extraction: a comparative study by R. A. Pérez; B. Albero; E. Miguel; C. Sánchez-Brunete (pp. 2347-2357).

Two rapid methods were evaluated for the simultaneous extraction of seven parabens and two alkylphenols from soil based on matrix solid-phase dispersion (MSPD) and microwave-assisted extraction (MAE). Soil extracts were derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide and analyzed by gas chromatography with mass spectrometry. Extraction and clean-up of samples were carried out by both methods in a single step. A glass sample holder, inside the microwave cell, was used in MAE to allow the simultaneous extraction and clean-up of samples and shorten the MAE procedure. The detection limits achieved by MSPD were lower than those obtained by MAE because the presence of matrix interferences increased with this extraction method. The extraction yields obtained by MSPD and MAE for three different types of soils were compared. Both procedures showed good recoveries and sensitivity for the determination of parabens and alkylphenols in two of the soils assayed, however, only MSPD yielded good recoveries with the other soil. Finally, MSPD was applied to the analysis of soils collected in different sites of Spain. In most of the samples analyzed, methylparaben and butylparaben were detected at levels ranging from 1.21 to 8.04 ng g⁻¹ dry weight and 0.48 to 1.02 ng g⁻¹ dry weight, respectively.

Keywords: GC-MS; MSPD; MAE; Alkylphenols; Parabens; Soil

Environmental analysis of alcohol ethoxylates and nonylphenol ethoxylate metabolites by ultra-performance liquid chromatography–tandem mass spectrometry by Pablo A. Lara-Martín; Eduardo González-Mazo; Bruce J. Brownawell (pp. 2359-2368).

Surfactants and their metabolites can be found in aquatic environments at relatively high concentrations compared with other micropollutants due in part to the exceptionally large volumes produced every year. We have focused our attention here on the most widely used nonionic surfactants, alcohol ethoxylates (AEOs), and on nonylphenol ethoxylate (NPEO) degradation products (short-chain nonylphenol ethoxylates, NP1-3EO, nonylphenol, NP, and nonylphenol ethoxycarboxylates, NP1-2EC), which are endocrine-disrupting compounds. Our main objective in this work was to develop a methodology aimed at the extraction, isolation, and improved analysis of these analytes in environmental samples at trace levels. Extraction recoveries of target compounds were determined for sediment samples after ultrasonic extraction and purification using HLB or C18 solid-phase extraction minicolumns. Recovery percentages were usually between 61 and 102% but were lower for longer AEO ethoxymers. Identification and quantification of target compounds was carried out using a novel ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC–MS-MS) approach, a combination that provides higher sensitivity and faster analysis than prior methods using conventional high-performance liquid chromatography–mass spectrometry. Limits of detection were usually below 0.5 ng/g, being higher for monoethoxylate species (>5 ng/g) because of poor ionization. The method was used for analyzing surface sediment samples collected at Jamaica Bay (NY) in 2008. The highest values (28,500 ng/g for NP, 4,200 ng/g for NP1-3EO, 22,400 ng/g for NP1-2EC, and 1,500 ng/g for AEOs) were found in a sampling station from a restricted water circulation area that is heavily impacted by wastewater discharges.

Keywords: Tandem mass spectrometry; Solid-phase extraction; Ultra-performance liquid chromatography; Sediments; Nonionic surfactants; Endocrine disruptor compounds

Automated analysis of perfluorinated compounds in human hair and urine samples by turbulent flow chromatography coupled to tandem mass spectrometry by Francisca Perez; Marta Llorca; Marinella Farré; Damià Barceló (pp. 2369-2378).

Perfluorinated compounds (PFCs) are ubiquitous contaminants of humans and animals worldwide. PFCs are bioaccumulated because of their affinity for proteins. It has been shown they could have a variety of toxicological effects and cause damage to human health, emphasizing the need for sensitive and robust analytical methods to assess their bioaccumulation in humans. In this paper we report the development and validation of an analytical method for analysis of PFCs in the non-invasive human matrices hair and urine. The method is based on rapid and simple sample pre-treatment followed by online turbulent flow liquid chromatography and tandem mass spectrometry (TFC–LC–MS–MS) for analysis of 21 PFCs. The method was validated for both matrices. Percentage recovery was between 60 and 105 for most compounds in both matrices. Limits of quantification ranged from 0.1 to 9 ng mL⁻¹ in urine and from 0.04 to 13.4 in hair. The good performance of the method was proved by investigating the presence of selected PFCs in 24 hair and 30 urine samples from different donors living in Barcelona (NE Spain). The results were indicative of bioaccumulation of these compounds in both types of sample. PFOS and PFOA were most frequently detected in hair and PFBA in urine.

Keywords: Perfluorinated compounds; LC–MS–MS; Turbulent flow chromatography; Hair; Urine

Probe functionalization with a Rhop-3 antibody: toward a Rhop-3 antigen immunosensor for detection of malaria by Salaam Saleh; Susan Moreno-Molek; Indika Perera; Alan Riga; Tobili Sam-Yellowe; Mekki Bayachou (pp. 2379-2384).

The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 °C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K₃Fe(CN)₆, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 °C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker.

Keywords: Immunosensor; Antibody immobilization; Malaria diagnosis; Plasmodium falciparum ; Rhop-3; Rapid diagnostic test

Glucose sensitive poly (N-isopropylacrylamide) microgel based etalons by Courtney D. Sorrell; Michael J. Serpe (pp. 2385-2393).

Thermoresponsive microgels have been shown to be an excellent platform for designing sensor materials. Recently, poly (N-isopropylacrylamide)-co-acrylic acid (pNIPAm-co-AAc) microgel based etalon materials have been described as direct sensing materials that can be designed to have a single, unique color. These color tunable materials show immense promise for sensing due to their spectral sensitivity and bright visual color. Here, we describe a proof-of-concept for etalon sensing of glucose. We found that aminophenylboronic acid (APBA)-functionalized pNIPAm-co-AAc microgels in an etalon respond to 3 mg/mL glucose concentrations by red shifting their reflectance peaks by 110 nm up to 150 nm. Additionally, APBA-functionalized pNIPAm-co-AAc microgels have a depressed volume phase transition temperature at 18–20 °C, which shifts to 24–26 °C after glucose binding. We also demonstrate that these materials show a marked visual color change, which is a first step towards developing direct read-out sensor devices. Figure Glucose sensitive, pNIPAm microgel-based etalons exhibit both a shift of the peaks in the reflectance spectrum, and a visual color change, upon exposure to biologically relevant concentrations of glucose.

Keywords: Poly (N-isopropylacrylamide) microgels; Color tunable materials; Fabry–Pérot etalon; Stimuli responsive polymers; Metal–polymer hybrid structures; Glucose sensing

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography by Jens Sproß; Andrea Sinz (pp. 2395-2405).

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics.

Keywords: Monolithic column; Affinity purification; Avidin; Biotin; Protein identification

Particle size measurement of lipoprotein fractions using diffusion-ordered NMR spectroscopy by Roger Mallol; Miguel A. Rodríguez; Mercedes Heras; Maria Vinaixa; Núria Plana; Lluís Masana; Gareth A. Morris; Xavier Correig (pp. 2407-2415).

The sizes of certain types of lipoprotein particles have been associated with an increased risk of cardiovascular disease. However, there is currently no gold standard technique for the determination of this parameter. Here, we propose an analytical procedure to measure lipoprotein particles sizes using diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY). The method was tested on six lipoprotein fractions, VLDL, IDL, LDL₁, LDL₂, HDL₂, and HDL₃, which were obtained by sequential ultracentrifugation from four patients. We performed a pulsed-field gradient experiment on each fraction to obtain a mean diffusion coefficient, and then determined the apparent hydrodynamic radius using the Stokes–Einstein equation. To validate the hydrodynamic radii obtained, the particle size distribution of these lipoprotein fractions was also measured using transmission electron microscopy (TEM). The standard errors of duplicate measurements of diffusion coefficient ranged from 0.5% to 1.3%, confirming the repeatability of the technique. The coefficient of determination between the hydrodynamic radii and the TEM-derived mean particle size was r ² = 0.96, and the agreement between the two techniques was 85%. Thus, DOSY experiments have proved to be accurate and reliable for estimating lipoprotein particle sizes. Figure

Keywords: Lipoprotein; NMR; DOSY; TEM

Inhibition of bacterial degradation of EtG by collection as dried urine spots (DUS) by Ana Hernández Redondo; Christiane Körber; Stefan König; Andreas Längin; Ali Al-Ahmad; Wolfgang Weinmann (pp. 2417-2424).

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers widely used nowadays for clinical and forensic applications. They are detectable in blood and urine even after consumption of trace amounts of ethanol and for a longer time frame, being detectable even when no more ethanol is present. The instability of EtG against bacterial degradation in contaminated urine samples and/or the possible postcollection synthesis of this metabolite in samples containing, e.g., Escherichia coli and ethanol, may cause false identification of alcohol uptake. Therefore, it is of paramount importance to constrict these error sources by inhibition of any bacterial growth causing hydrolization or synthesis of EtG. This study evaluates a new method of collecting urine samples on filter paper, dried urine spots (DUS), for simultaneous detection of EtG, EtS and creatinine, having the great advantage of inhibiting bacterial activity. In addition, a method validation for the determination of EtG and EtS in DUS was performed according to the FDA guidelines. Sterile-filtered urine was spiked with EtG and EtS, inoculated with E. coli and incubated. Liquid and dried urine samples were collected after various time intervals up to 96 h. Liquid samples were frozen immediately after collection, whereas aliquots for DUS were pipetted onto filter paper, allowed to dry and stored at RT until analysis 1 week after. The specimens were analyzed by LC–ESI–MS/MS. As expected, degradation of EtG, but not of EtS, was observed in contaminated liquid urine samples. However, the specimens collected on filter paper and stored at RT showed no degradation during storage. Therefore, collecting urine samples on filter paper for EtG and EtS analysis turns out to be a reliable method to avoid bacterial degradation of EtG and EtS, and consequently, stabilization of these ethanol metabolites is achieved. In addition, simultaneous measurement of creatinine content as an indicator of urine dilution helps to interpret the results. Method validation for EtG and EtS in DUS was satisfactory, showing the linearity of the calibration curves in the studied concentration range, good precision, accuracy and selectivity.

Keywords: Ethyl glucuronide (EtG); Ethyl sulfate (EtS); Bacterial degradation; Dried urine spot (DUS); Creatinine; LC–ESI–MS/MS

Capillary ion electrophoresis–capacitively coupled contactless conductivity detection of inorganic cations in human saliva on a polyvinyl alcohol-coated capillary by Masanobu Mori; Maki Kaseda; Tsukasa Yamamoto; Sachiko Yamada; Hideyuki Itabashi (pp. 2425-2430).

Capillary ion electrophoresis–capacitively coupled contactless conductivity detection (CIE-C4D) with a polyvinyl alcohol chemically coated capillary (PVA capillary) was used to analyze inorganic cations (Na⁺, K⁺, NH ₄ ⁺ , Mg²⁺, and Ca²⁺) commonly found in human saliva. The PVA capillary, which was made by our laboratory, minimized electro-osmotic flow in the wide pH range of the background electrolyte (BGE), and the PVA layer adsorbed to capillary wall did not affect the conductimetric background level. In this study, we determined an optimized BGE of 30 mM lactic acid/histidine plus 3 mM 18-crown-6 for the CIE-C4D system using the PVA capillary, which could simultaneously improve the separation of Mg²⁺ and Ca²⁺ from Na⁺ and that of K⁺ from NH ₄ ⁺ . This system obtained highly reproducible separation of cations in human saliva samples within 8 min at 20 kV without deprotonation. The quantifiability of cations in human saliva samples on the CIE-C4D system was demonstrated through identification by ion chromatography with satisfactory results.

Keywords: Capillary ion electrophoresis; Capacitively coupled contactless conductivity; PVA-coated capillary; Human saliva; Inorganic cations

Determination of 2,3-dihydroxypropionamide, an oxidative metabolite of acrylamide, in human urine by gas chromatography coupled with mass spectrometry by Julia M. Latzin; Birgit K. Schindler; Tobias Weiss; Jürgen Angerer; Holger M. Koch (pp. 2431-2438).

The general population is exposed to acrylamide (AA) mainly through food and tobacco smoke. AA is classified as probably carcinogenic to humans. Glycidamide (GA), as the primary oxidative metabolite, was identified to be the ultimate genotoxic agent. This warrants full investigation of the oxidative pathway in AA metabolism and the share of the oxidative compared to the reductive pathway. 2,3-Dihydroxy-propionamide (OH-PA) as the direct hydrolysis product of GA has been shown to be a major urinary oxidative metabolite in human AA metabolism. We developed an analytical method to reliably quantify OH-PA in urine by GC-MS after a multistep procedure including “stripping” on a solid phase material, lyophilization, silylation and re-extraction. With a detection limit of 1 μg/L, our method is sensitive enough to quantify OH-PA in all urine samples of the general population. Within and between series precisions were between 1.9% and 8.2% and mean recoveries between 97% and 101%. We applied this method to 30 urine samples from the general population. In all the samples, OH-PA was present in concentrations between 6.8 and 109.4 μg/L (median, 49.7 μg/L) with no difference between smokers and non-smokers. OH-PA concentrations were approximately ten times higher than expected from the metabolism of AA via GA. Currently, we cannot confirm OH-PA to be a specific biomarker of the oxidative pathway of AA metabolism. Other sources than AA respectively GA might need to be considered for the formation of OH-PA.

Keywords: Acrylamide (AA); Glycidamide (GA); Biological monitoring; 2,3-Dihydroxy-propionamide (OH-PA); GC/MS; Urine

Quantification of femtomolar concentrations of the CYP3A substrate midazolam and its main metabolite 1′-hydroxymidazolam in human plasma using ultra performance liquid chromatography coupled to tandem mass spectrometry by Jürgen Burhenne; Birte Halama; Monika Maurer; Klaus-Dieter Riedel; Nicolas Hohmann; Gerd Mikus; Walter E. Haefeli (pp. 2439-2450).

The benzodiazepine midazolam is a probe drug used to phenotype cytochrome P450 3A activity. In this situation, effective sedative concentrations are neither needed nor desired, and in fact the use of very low doses is advantageous. We therefore developed and validated an assay for the femtomolar quantification of midazolam and 1′-hydroxymidazolam in human plasma. Plasma (0.25 mL) and 96-well-based solid-phase extraction were used for sample preparation. Extraction recoveries ranged between 75 and 92% for both analytes. Extracts were chromatographed within 2 min on a Waters BEH C18 1.7 μm UPLC® column with a fast gradient consisting of formic acid, ammonia, and acetonitrile. Midazolam and 1′-hydroxymidazolam were quantified using deuterium- and ¹³C-labeled internal standards and positive electrospray tandem mass spectrometry in the multiple reaction monitoring mode, which yielded lower limits of quantification of 50 fg/mL (154 fmol/L) and 250 fg/mL (733 fmol/L) and a corresponding precision of <20%. The calibrated concentration ranges were linear for midazolam (0.05–250 pg/mL) and 1′-hydroxymidazolam (0.25–125 pg/mL), with correlation coefficients of >0.99. Within-batch and batch-to-batch precision in the calibrated ranges for both analytes were <14% and <12%. No ion suppression was detectable, and plasma matrix effects were minimized to <15% (<25%) for midazolam (1′-hydroxymidazolam). The assay was successfully applied to assess the kinetics of midazolam in two human volunteers after the administration of single oral microgram doses (1–100 μg). This ultrasensitive assay allowed us to quantify the kinetics of midazolam and 1′-hydroxymidazolam for at least 10 h, even after the administration of only 1 μg of midazolam. Figure UPLC/MS/MS quantification of femtomolar midazolam plasma concentrations

Keywords: Midazolam; 1′-hydroxymidazolam; UHPLC; Mass spectrometry; CYP3A

Determination of 16 insect growth regulators in edible Chinese traditional herbs by liquid chromatography electrospray tandem mass spectrometry by Mingrong Qian; Liqin Wu; Hu Zhang; Mingfei Xu; Rui Li; Xiangyun Wang; Caixia Sun (pp. 2451-2462).

A new sensitive multiresidue liquid chromatography–tandem mass spectrometry (LC-MS/MS) analytical method for the determination of 16 insect growth regulator (IGR) residues—RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), halofenozide, methoxyfenozide, chromafenozide, fufenozide, tebufenozide, diflubenzuron, chlorbenzuron, triflumuron, hexaflumuron, novaluron, lufenuron, teflubenzuron, flucycloxuron, flufenoxuron, and chlorfluazuron—in herbs (Perilla frutescens, flos chrysanthemi, lily bulbs, and ginger) has been developed. After the herbs had been extracted with acetonitrile, a combined graphitized nonporous carbon/aminopropyl (ENVI-Carb/LC-NH₂) cartridge and a Florisil cartridge were used to clean up the extracts. LC-MS/MS was performed in multiple reaction monitoring mode with two specific precursor ion–product ion transitions per IGR to confirm and quantitate the residues in herbs. Quantitation was performed on the basis of matrix-matched calibrations. The method showed excellent linearity (r ² > 0.99) and precision (relative standard deviations of 13.6 or lower) for all the target insecticides. The limits of quantitation were 0.6-10 μg kg^-1 for the 16 insecticides in the four herbs. The average recoveries, measured at three concentrations (0.01, 0.1, 1 mg kg^-1), were in the range 74.8-105.3%. The method was satisfactorily applied for the analysis of 60 herb samples (Perilla frutescens, flos chrysanthemi, lily bulbs, and ginger). Hexaflumuron was detected at concentrations of 0.029 and 0.051 mg kg^-1 in Perilla frutescens. Figure The chromatograms for LC-MS/MS of 16 insect growth regulators

Keywords: Residue analysis; Insect growth regulators; High-performance liquid chromatography–tandem mass spectrometry; Diacylhydrazine; Benzoylphenylurea; Herbs

A Monte Carlo approach for estimating measurement uncertainty using standard spreadsheet software by Gina Chew; Thomas Walczyk (pp. 2463-2469).

Despite the importance of stating the measurement uncertainty in chemical analysis, concepts are still not widely applied by the broader scientific community. The Guide to the expression of uncertainty in measurement approves the use of both the partial derivative approach and the Monte Carlo approach. There are two limitations to the partial derivative approach. Firstly, it involves the computation of first-order derivatives of each component of the output quantity. This requires some mathematical skills and can be tedious if the mathematical model is complex. Secondly, it is not able to predict the probability distribution of the output quantity accurately if the input quantities are not normally distributed. Knowledge of the probability distribution is essential to determine the coverage interval. The Monte Carlo approach performs random sampling from probability distributions of the input quantities; hence, there is no need to compute first-order derivatives. In addition, it gives the probability density function of the output quantity as the end result, from which the coverage interval can be determined. Here we demonstrate how the Monte Carlo approach can be easily implemented to estimate measurement uncertainty using a standard spreadsheet software program such as Microsoft Excel. It is our aim to provide the analytical community with a tool to estimate measurement uncertainty using software that is already widely available and that is so simple to apply that it can even be used by students with basic computer skills and minimal mathematical knowledge.

Keywords: Metrology; Monte Carlo; Measurement uncertainty; Spreadsheet

Simultaneous determination of benzotriazole and benzothiazole derivatives in aqueous matrices by mixed-mode solid-phase extraction followed by liquid chromatography–tandem mass spectrometry by I. Carpinteiro; B. Abuin; M. Ramil; I. Rodríguez; R. Cela (pp. 2471-2478).

An improved selectivity method for the simultaneous determination of four benzotriazoles (benzotriazole, 4-methylbenzotriazole, 5-methylbenzotriazole, and 5,6-dimethyl-1H-benzotriazole) and six benzothiazoles (benzothiazole, 2-hydroxybenzothiazole, 2-benzothiazolamine, mercaptobenzothiazole, 2-methylbenzothiazole, and 2-methylthiobenzothiazole) in aqueous matrices has been developed. Under optimal conditions, analytes are concentrated using a MAX solid-phase extraction (SPE) cartridge, based on divinylbenzene-N-vinylpyrrolidone functionalized with quaternary amine groups, which allows reversed-phase interactions in combination with ionic exchange. Selected compounds are recovered with methanol–acetone 7:3 (v/v) whereas acidic interferences remained attached to the sorbent, and as determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), LOQs for surface, urban and industrial wastewater are in the range of 0.002–0.29 ng/mL. Figures of merit of the method revealed good precision (RSD% <12%), linearity (R ² > 0.99) and accuracy (%R = 80–100%) for surface waters and effluents allowing direct external standard quantification. For more complex samples, such as urban and industrial raw wastewater, either the standard addition method or pseudo-external standard calibration using matrix matched standards are recommended. Analysis of different real samples, surface, urban wastewater and, for the first time, metal industry wastewater, reflected concentrations up to 310 ng/mL. The methylbenzotriazole isomers ratio was also determined.

Keywords: Benzotriazoles; Benzothiazoles; Water analysis; Mixed-mode solid-phase extraction; Liquid chromatography coupled to tandem mass spectrometry; Emerging contaminants

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Analytical and Bioanalytical Chemistry (v.402, #7)

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Analytical and Bioanalytical Chemistry (v.402, #7)